Novel variants of RANKL protein

ABSTRACT

The present invention relates to novel, soluble, recombinant variants RANKL (Receptor Activator of Nuclear Factor—κB Ligand) proteins, which may be expressed solubly in  E. coli,  variants that act as RANKL antagonists, and methods for generating the same.

[0001] This application claims the benefit of the filing date of U.S.Ser. No. 60/345,805, filed Jan. 4, 2002 and U.S. Ser. No. 60/373,453,filed Apr. 17, 2002, all of which are expressly incorporated byreference in their entirety.

[0002] The present application is related to a co-pending applicationfiled on even date and entitled “DOMINANT NEGATIVE PROTEINS AND METHODSTHEREOF, hereby incorporated by reference in its entirety.”

FIELD OF THE INVENTION

[0003] The present invention relates to novel variants of theextracellular domains (ECD) of human RANKL (Receptor Activator ofNuclear Factor—κB Ligand) proteins and fragments, derivatives,conformers, and analogs thereof. These novel variants comprise RANKLvariants that express solubly in E. coli, RANKL variants that antagonizewild type RANKL, and RANKL variants that act as superagonists.

BACKGROUND OF THE INVENTION

[0004] Normal bone remodeling is a process in which new bone depositionby osteoblasts is balanced through bone resorption by osteoclasts (seeGowen, M., Every, J G, and Kumar S. Emerging therapies for osteoporosis.Emerging Drugs, 2000 5(1): p.1-43.) In several disease states, thebalance between bone deposition and bone resorption is perturbed. Inosteoporosis, for example, excess bone resorption leads to brittle bonesand frequent fractures of the wrist, vertebrae, and hip. In rheumatoidarthritis, increased bone resorption leads to malformations of the boneswithin arthritic joints. Re-establishing normal bone remodeling in theseand other disorders can be achieved by decreasing or increasing thenumber and activity of osteoclasts (See Rodan, G A, and Martin T J.Therapeutic approaches to bone disease. Science 2000 289: p. 1508-1514.)

[0005] Several proteins modulate the bone remodeling orchestrated byosteoblasts and osteoclasts. Three key proteins are the cell-surfacereceptor RANK (Receptor Activator of NF-κB), the soluble decoy receptorOPG (osteoprotegerin), and the soluble and transmembrane forms of RANKL(RANK ligand, also known as RANKL11, TNF-related activation inducedcytokine (TRANCE), osteoclast differentiation factor (ODF), andosteoprotegerin ligand (OPGL)). RANK is activated by the binding of itsligand, RANKL, which leads to the differentiation, survival, and fusionof pre-osteoclasts to form active bone resorbing osteoclasts (see LaceyD L, Timms E, Tan H-L, Kelley M J, Dunstan C R, Burgess T et al.Osteoprotegerin Ligand is a cytokine that regulates osteoclastdifferentiation and activation. 1998 Cell 93: p. 165-176.). RANKL is atrimeric TNF family member that binds to the trimeric RANK receptor.

[0006] The RANKL/OPG/RANK biochemical axis has been successfullytargeted to treat osteoporosis, rheumatoid arthritis, cancer-inducedbone destruction, metastasis, hypercalcemia, and pain (see Hofbauer, LC, Neubauer, A, and Heufelder A E. Receptor activator of nuclearfactor-κB ligand and osteoprotegrin. 2001 Cancer 92(3): p.460-470;Takahashi N, Udagawa N, and Suda T.) Therapies utilizing OPG (see HonoreP, Luger N M, Samino M A C, Schwei M J et al. Osteoprotegerin blocksbone cancer-induced skeletal destruction, skeletal pain and pain-relatedneurochemical reorganization of the spinal cord. 2000 Nature Medicine6(5):521-528.) or the soluble RANK-Fc protein (See Oyajobi B O, AndersonD M, Traianedes K, Williams P J, Yoneda T, Mundy G R. Therapeuticefficacy of a soluble receptor activator of nuclear factor kappaB-IgG Fcfusion protein in suppressing bone resorption and hypercalcemia in amodel of humoral hypercalcemia of malignancy. 2001 Cancer Res 61(6): p.2572-8) are also in development. OPG and soluble RANK-Fc proteinconstructs bind to RANKL, thereby decreasing amount of RANKL that isavailable for RANK receptor activation.

[0007] Hypercalcemia is a late stage complication of cancer, disruptingthe body's ability to maintain normal levels of calcium, resulting incalcium deposit in the kidneys, heart conditions and neural dysfunctionand occurs most frequently in patients cancers of the with lung andbreast. Hypercalcemia also occurs in patients with multiple myeloma,cancers of the head and neck, sarcoma, cancers of unknown primaryorigin, lymphoma, leukemia, melanoma, renal cancer, and gastrointestinalcancers (e.g. esophageal, stomach, intestinal, colon and rectalcancers).

[0008] In addition to being important in bone biology, RANKL plays arole in the immune system by regulating antigen-specific T cellresponses (See Anderson et al., A homologue of the TNF receptor and itsligand enhance T-cell growth and dendritic-cell function; Nature 1997,390(6656):175-9). RANKL is highly expressed on activated T cells whilethe RANK receptor is expressed at high levels on mature dendritic cells(DC). The interaction between RANKL and RANK acts as a costimulatorysignal, which enhances DC survival and T cell proliferation by inducingDC differentiation, cytokine production and reduced apoptosis in bothcell types. Immunotherapy to produce tolerance to transplanted tissuesand/or organs can be achieved by blocking the costimulatory signal usingRANK antagonists. Blocking costimulation prevents T cell activation byDCs, and causes alloreactive T cells to become anergic and/or undergoapoptosis (See Adler et al., Immunotherapy as a means to inducetransplantation tolerance; Current Opinion in Immunology 2002,14:660-665). By a similar mechanism of action, antagonizing RANKsignaling could be a treatment for autoimmune disorders such as systemiclupus erythematosus, inflammatory bowel disease, diabetes, multiplesclerosis, rheumatoid arthritis, and ankylosing spondylitis. RANKLvariant superagonists, on the other hand could be used to activate theimmune system by promoting T cell activation. RANKL superagonists couldbe useful treatments for diseases including but not limited to cancerand viral infection.

[0009] Much work has been done to develop therapeutic entities andreagents for biological research based on RANKL. For example, RANKLfragments, analogs, derivatives, or conformers having the ability tobind OPG, which could be used as treatments for a variety of bonediseases, have been described (See U.S. Pat. No. 5,843,678). RANKLvariants, which induce production of an immune response thatdown-regulates RANKL activity, have been disclosed (See WO 00/15807). Inother studies, utilization of RANKL protein and its derivatives asimmune modulators has been proposed (See WO 99/29865). All referencescited herein are hereby expressly incorporated by reference.

[0010] Accordingly, a need exists for RANKL antagonists andsuperagonists.

SUMMARY OF THE INVENTION

[0011] The present invention is directed at generating novel variants ofhuman RANKL protein, comprising the extracellular domains of RANKL,which behave as RANKL antagonists or superagonists. Here, we describenovel variants of human RANKL protein that behave as RANKL antagonistsand superagonists. RANKL antagonists may comprise dominant-negativeRANKL proteins and/or competitive inhibitors of RANKL. A furthercomponent of the invention is the identification of modifications thatconfer soluble expression in E. coli. Soluble expression allows forefficient and cost-effective production and manufacturing of human RANKLvariants, and therefore is critical for the discovery, characterization,and production of novel RANKL antagonists and superagonists.

[0012] An aspect of the present invention is non-naturally occurringRANKL variants that express solubly in bacteria, including but notlimited to E. coli. In previous studies, it has been observed that humanRANKL forms inclusion bodies when expressed in E. coli. Alternateproduction routes such as refolding from inclusion bodies or mammalianexpression are significantly more expensive and time consuming thansoluble bacterial expression. Soluble bacterial expression facilitatesthe discovery, characterization, and production of novel RANKL variants.It is a further object of the invention to provide a method that can beused to engineer variants of other proteins that express solubly inbacteria, including but not limited to E. coli.

[0013] The invention further relates to the design of human RANKLantagonists that inhibit the interaction between RANK receptor and RANKLand methods for generating the same.

[0014] An aspect of the present invention is non-naturally occurringRANKL variants that: 1) do not appreciably agonize RANK activity; 2)antagonize RANK activity; 3) exchange with wild-type RANKL (that is,form trimers containing at least one wild type RANKL protein monomer andat least one variant RANKL protein monomer), and 4) interfere with RANKmediated osteoclast formation and/or T cell costimulation. Such variantsare referred to as “dominant negative” variants.

[0015] A further object of the present invention is RANKL variants thatpreferentially heterotrimerize with wild-type RANKL.

[0016] An aspect of the present invention is RANKL variants that: 1) donot appreciably agonize RANK activity; 2) antagonize RANK activity; 3)compete with wild type RANKL for binding the RANK receptor, and 4)interfere with RANK mediated osteoclast formation and activation and/orT cell costimulation. Such variants are referred to as “competitiveinhibitor” variants.

[0017] The invention further relates to the design of human RANKLsuperagonists that bind and activate the RANK receptor more stronglythan the wild type human RANKL protein does.

[0018] In a further aspect, the invention provides recombinant nucleicacids encoding the variant RANKL proteins, expression vectors, and hostcells.

[0019] In an additional aspect, the invention provides methods ofproducing a variant RANKL protein comprising culturing the host cells ofthe invention under conditions suitable for expression of the variantRANKL protein.

[0020] In a further aspect, the invention provides pharmaceuticalcompositions comprising a variant RANKL protein of the invention and apharmaceutical carrier.

[0021] In a further aspect, the invention provides methods for treatingRANKL-related disorders comprising administering a variant RANKL proteinof the invention to a patient.

[0022] In accordance with the objects outlined above, the presentinvention provides RANKL variant proteins comprising amino acidsequences with at least one amino acid change compared to the wild typeRANKL proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1a shows the amino acid sequence of wild-type human RANKLextracellular region (amino acids 68-317 of GenBank accession AAB86811;SEQ ID NO: 1).

[0024]FIG. 1b shows the amino acid sequence of wild-type human RANKLextracellular region with 6× histidine tag and TEV protease cleavagesite. This construct and variants thereof were expressed and assayedbiochemically and functionally in cell-based assays (SEQ ID NO: 2)

[0025]FIG. 1c shows the amino acid sequence of wild-type mouse RANKLextracellular region (amino acids 68-317 of GenBank accessionNP_(—)035743; SEQ ID NO: 3).

[0026]FIG. 2a shows a BLAST alignment between mouse and human wild-typeRANKL protein sequences.

[0027]FIG. 2b shows a listing of mutations of several human RANKLsolubility variants.

[0028]FIG. 3 shows the strategy for generating single chaindominant-negative RANKL variants. The linked dimer forms an inactivecomplex with wild-type RANKL. Through this mechanism of inactivatingwild-type RANKL, the single chain dominant-negative RANKL variants willexert their therapeutic effects.

[0029]FIG. 4 shows expression analysis that demonstrates soluble E. coliexpression of C221S/I207R and C221S/I247E (arrows). RANKL variantsC221S/173E, C221S/173R, C221S/S183R, C221S/A232K and C221S/I207E do notexhibit soluble expression. Wild-type RANKL was also insoluble (data notshown). M is molecular weight marker; U is uninduced bacterial lysate, Wis whole cell bacterial lysate, and S is soluble fraction

[0030]FIG. 5 shows expression analysis that demonstrates soluble E. coliexpression of the additional RANKL variants C221S/I207K, C221S/I247K,and C221S/I247E and demonstrates lack of soluble E. coli expression ofthe variant C221S/I207D. M is molecular weight marker; U is uninducedbacterial lysate, T is whole cell bacterial lysate, and S is solublefraction.

[0031]FIG. 6 shows expression analysis that demonstrates soluble E. coliexpression of RANKL variants C221S/I207K and C221S/I247E. Wild-typeRANKL is not soluble along with several other variants.

[0032]FIG. 7 shows that the C221S mutation when combined with the I247Emutation confers soluble RANKL expression in E. coli.

[0033]FIG. 8 shows that the C221S mutation when combined with the I247E,I247K, or I247Q mutations confer soluble expression in E. coli. Each ofthe single mutations in isolation is not sufficient to confer solubleexpression.

[0034]FIG. 9 shows Ni NTA batch purification from soluble fraction ofthe two RANKL variants C221S/I247E and C221S/I247D. For eachpurification, fractions from lanes 11-13 were pooled and equilibriatedin PBS, pH 8.0. Analysis shows that soluble variants can be expressed tohigh levels and purified.

[0035]FIG. 10 shows a quality control gel of highly purified solubleRANKL protein preparations from the six variants C221S/I247E,C221S/I247D, C221S/I247K, C221S/I247Q, C221S/I247A, and C221S/I207K .

[0036]FIG. 11 shows light scattering and size exclusion chromatographydata indicating that solubility variant C221S/I247E is 97% trimeric.This analysis demonstrates that the variant protein is folded properlyand forms a trimer as is expected for wild-type RANKL protein.

[0037]FIG. 12 Summary of light scatter-size exclusion chromatographyshowing that all six human RANKL variants are predominantly timeric.Expression levels are expressed as mass of RANK protein purified permass of bacteria producing the protein.

[0038]FIG. 13 shows Western analysis indicating that the 6 RANKLsolubility variants bind the human receptor RANK in the context of aRANK-Fc fusion construct. The ability of the RANKL variants to bind RANKwas assessed in a receptor precipitation experiment. 200 ng of Histagged RANKL was incubated with 1 μg of RANK-Fc for 1 hour at 4° C. inDMEM/10%FBS/10 mM Hepes/0.01% Tween 20. Protein A/G beads were added andallowed to incubate on ice for an additional 1 hr. Immune complexes werecollected by centrifugation and washed twice with binding buffer andthen once with PBS. Samples were analyzed by SDS-PAGE followed byimmunoblotting with an anti-His antibody. His-tagged TNFα was capturedby TNFR1-Fc as a positive control and His-tagged TNFα incubated withRANK-Fc acted as the negative control.

[0039]FIG. 14 shows Western analysis indicating that the six RANKLsolubility variants bind human OPG-Fc. The ability of variant RANKL tobind OPG was assessed in a immuno-precipitation experiment. 200 ng ofHis tagged RANKL was incubated with 1 μg of OPG-Fc for 1 hour at 4° C.in DMEM/10% FBS/10 mM Hepes/0.01% Tween 20. Protein A/G beads were addedand allowed to incubate on ice for an additional 1 hr. Immune complexeswere collected by centrifugation and washed twice with binding bufferand then once with PBS. Samples were analyzed by SDS PAGE followed byimmunoblotting with an anti-RANKL antibody.

[0040]FIG. 15 shows TRANSFACTOR™ (commercially available from Clontech)analysis demonstrating that the RANKL variant (C221S/I247E) activatesthe NFκB pathway. This analysis indicates that the solubility variant isfunctionally active.

[0041]FIG. 16 shows TRANSFACTOR™ (commercially available from Clontech)analysis demonstrating that the RANKL variant (C221S/I247E) activatesthe JNK pathway. This analysis indicates that the solubility variant isfunctionally active.

[0042]FIG. 17 shows that the RANKL solubility variant C221S/I247Einduces osteoclastogenesis in mouse RAW264.7 cells. RAW264.7 cells wereplated and incubated with RANKL (C221S/I247E) at the indicated doses for3 days. Tartrate resistant acid phosphatase (TRAP) staining revealedmultinucleated osteoclast cells in a dose-dependant manner.

[0043]FIG. 18 shows that RANKL solubility variant C221S/I247E inducesosteoclastogenesis in mouse RAW264.7 cells. RAW264.7 cells were platedand incubated with proteins at various doses for 3 days. Tartrateresistant acid phosphatase (TRAP) activity measurements usingp-nitrophenyl phosphate as substrate show that variant RANKL variantC221/I247E has similar activity to wild-type RANKL commerciallyavailable from Biosource Inc.

[0044]FIG. 19 shows that six RANKL solubility variants induceosteoclastogenesis in mouse RAW264.7 cells. RAW264.7 cells were platedand incubated with proteins at various doses for 3 days. Tartrateresistant acid phosphatase (TRAP) activity measurements usingp-nitrophenyl phosphate as substrate show that the RANKL variants areail active. Human and mouse wild-type RANKL from R&D Systems areincluded as controls.

[0045]FIG. 20 shows that osteoclastogenesis mediated by RANKL solubilityvariant C22S/I247E is antagonized by OPG and RANK-Fc. The plots showthat commercial RANKL from BioSource is antagonized to the same extentas Xencor solubility variant. The BSA and ENBREL® controls show that theantagonism is specific to RANKL interacting with OPG and RANK-Fc. TheRANK-Fc and OPG controls show that the variants do not mediateosteoclastogenesis.

[0046]FIG. 21 shows a model for the dominant-negative inhibitory RANKLvariant strategy. The RANKL variant homotrimers and heterotrimers withwild-type RANKL do not activate the RANK receptor. The RANKL variantsact in a dominant-negative manner by rendering the wild-type RANKLprotein inactive through the formation of these heterotrimer species.The bumps and sticks on the engineered RANKL protein symbolize therationally designed mutations in the small and large binding domains ofRANKL that interfere with receptor binding. Through this mechanism ofinactivating wild-type RANKL, the dominant-negative inhibitory RANKLvariants will exert their therapeutic effects.

[0047]FIG. 22 shows the list of computationally designed inhibitoryRANKL variants. These inhibitory variants can be combined with RANKLsolubility variants to produce soluble human RANKL variants.

[0048]FIG. 23. RANKL variant agonism screen. The ability of RANKLvariants to agonize osteoclastogenesis was evaluated by monitoring TRAPlevels. TRAP is released from RAW264.7 cells as they undergoRANK-mediated osteoclastogenesis. This experiment identified several nonagonizing RANKL variants and one superagonist (RANKL C221S/I247E/A172R).

[0049]FIG. 24. RANKL variant agonism screen. The ability of RANKLvariants to agonize osteoclastogenesis was evaluated by monitoring TRAPlevels. TRAP is released from RAW264.7 cells as they undergoRANK-mediated osteoclastogenesis. This experiment identified several nonagonizing RANKL variants.

[0050]FIG. 25. RANKL variant agonism screen. The ability of RANKLvariants to agonize osteoclastogenesis was evaluated by monitoring TRAPlevels. TRAP is released from RAW264.7 cells as they undergoRANK-mediated osteoclastogenesis. This experiment identified several nonagonizing RANKL variants.

[0051]FIG. 26. RANKL variant antagonism screen. The ability of RANKLvariants to antagonize osteoclastogenesis was evaluated by monitoringTRAP levels when RANKL-C221S/I247E is mixed with RANKL variants,incubated and added to RAW264.7 cells. TRAP is released from RAW264.7cells as they undergo RANK-mediated osteoclastogenesis. This experimentidentified RANKL variants that antagonized this process.

[0052]FIG. 27. RANKL variant antagonism screen using a fixed ratio ofRANKL-C221S/I247E to ten fold excess variants. The ability of RANKLvariants to antagonize osteoclastogenesis was evaluated by monitoringTRAP levels when RANKL-C221S/I247E is mixed with RANKL variants,incubated, diluted over 2 logs, and added to RAW264.7 cells. TRAP isreleased from RAW264.7 cells as they undergo RANK-mediatedosteoclastogenesis. This experiment identified RANKL variants thatantagonized this process.

[0053]FIG. 28 RANKL variant antagonism screen. The ability of RANKLvariants to antagonize osteoclastogenesis was evaluated by monitoringTRAP levels when RANKL-C221S/I247E and RANKL variants are added toRAW264.7 cells without pre-incubation. TRAP is released from RAW264.7cells as they undergo RANK-mediated osteoclastogenesis. This experimentidentified RANKL variants that antagonized this process.

[0054]FIG. 29 Summary of RANKL agonism screens. Twenty-one RANKLvariants were identified to be non-agonists while 12 were weak agonists.One RANKL variant, C221S/I247E/A172R is a superagonist.

[0055]FIG. 30 Summary of RANKL antagonizing variants. These variantswere shown to antagonize RANKL mediated osteoclastogenesis.Definitions

[0056] By “RANK” herein is meant a cell-surface receptor activator ofNF-κB. The RANK protein (or nucleic acid) can be from proteins may befrom any number of organisms, with RANK proteins from mammals beingparticularly preferred. Suitable mammals include, but are not limitedto, rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farmanimals (including sheep, goats, pigs, cows, horses, etc); and in themost preferred embodiment, from humans (the sequence of which isdepicted in the figures). As will be appreciated by those in the art,RANK proteins based on RANK proteins from mammals other than humans mayfind use in animal models of human disease.

[0057] By “RANKL” herein is meant a ligand for RANK. Again, RANKLsequences from humans are preferred (sequences depicted in the Figures),but RANKL sequences from organisms outlined above are also included. Asis more fully outlined below, RANKL proteins trimerize (although higheroligomers may also occur) to activate the RANK receptor. Thus, RANKLproteins occur as monomers as well as oligomers, generally trimers. Asmore fully outlined below, variant RANKL protein monomers can be madewhich physically interact with other monomers (including other variantRANKL monomers, wild-type monomers, or wild-type monomers from differentspecies) to form mixed oligomers or trimers.

[0058] By “variant RANKL monomer proteins” or grammatical equivalentsherein is meant a RANKL monomer protein that contains at least onemodification, including substitutions, insertions and deletions. Thus,variant RANKL proteins means non-naturally occurring RANKL proteins thatdiffer from the wild type RANKL protein by at least 1 modification,including but not limited to amino acid substitution, insertion, ordeletion. RANKL variants are characterized by the predetermined natureof the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the RANKL proteinsequence. The RANKL variants typically either exhibit the samequalitative biological activity as the naturally occurring RANKL or havebeen specifically engineered to have alternate biological properties.Variant RANKL proteins are derived from the extracellular domain of wildtype RANKL (that is, residues 68-317 in FIG. 1a). However, the variantRANKL proteins may contain insertions or deletions at the N-terminus,C-terminus, or internally. For example, a preferred truncated variant ofthe human RANKL protein comprises residues 159-317, as shown in FIG. 1b.Thus, in a preferred embodiment, variant RANKL proteins have at least 1residue that differs from the human RANKL sequence, with at least 2, 3,4, or 5 different residues being more preferred. Variant RANKL proteinsmay comprise one domain or multiple domains connected by linkersequences. Variant RANKL proteins may contain further modifications, forinstance modifications that alter stability or immunogenicity or whichenable posttranslational modifications such as PEGylation orglycosylation.

[0059] In general, deletions include portions of RANKL that are notextracellular, and include substitutions in residues corresponding tomonomer association sites and receptor recognition and/or binding sites.Preferred variants are further described below, and include bothconservative and non-conservative substitutions, with the latter beingpreferred.

[0060] By “extracellular domain” or “ECD” as used herein is meant thesegment of protein existing predominantly outside the cell. Fortransmembrane proteins, this segment can be tethered to the cell througha transmembrane domain or released from the cell through proteolyticdigestion. Alternatively, the extracellular domain could comprise thewhole protein or amino acid segments thereof when secreted from thecell. In general, RANKL members are expressed as type II transmembraneproteins (extracellular C terminus). Soluble forms of RANKL proteins mayresult from proteolytic cleavage of the signal propeptide by matrixmetalloproteinases termed TNF-alpha converting enzymes (TACE) ordirectly by recombinant methods. Unless otherwise disclosed, the variantRANKL proteins of the present invention are composed of theextracellular domain or functional equivalents thereof. That is, thevariants of the present invention do not comprise transmembrane domainsunless specifically noted.

[0061] By “agonists of wild-type RANKL” or grammatical equivalentsthereof, herein is meant variants of RANKL which themselves or incombination with wild type RANKL appreciably activate RANK receptor. Ina preferred embodiment, activation of the RANK receptor by agonisticRANKL variants is between 50% and 110% of the activation that would beattained with an equal amount of wild type RANKL protein, with between80% and 100% being especially preferred.

[0062] By “superagonists of wild-type RANKL” or grammatical equivalentsthereof, herein is meant variants of RANKL that enhance the activationof RANK receptor signaling by wild type RANKL proteins or thatthemselves activate the RANKL receptor more strongly than an equalamount of wild type RANKL. In a preferred embodiment the RANKLsuperagonist proteins yield at least 10% more activity than wild typeRANKL, with at least 50%, 100% or 200% increases in activity beingespecially preferred.

[0063] By “altered property” or grammatical equivalents thereof hereinis meant, in the context of a protein, any characteristic or attributeof a protein that differs from the corresponding property of a naturallyoccurring protein. These properties include, but are not limited tocytotoxic activity; oxidative stability, substrate specificity,substrate binding or catalytic activity, thermal stability, alkalinestability, pH activity profile, resistance to proteolytic degradation,kinetic association (Kon) and dissociation (Koff) rate, protein folding,immunogenicity, osteoclastogenesis, binding affinity and selectivity,ability to be secreted, ability to be displayed on the surface of acell, ability to oligomerize, ability to signal, ability to stimulatecell proliferation, ability to activate receptors; ability to inducedifferentiation, survival and fusion of pre-osteoclasts, ability toinhibit cell proliferation, ability to induce apoptosis, ability to bemodified by phosphorylation or glycosylation, and the ability to treatdisease. Unless otherwise specified, a property of a RANKL variant isconsidered to be “altered” when the property exhibits preferably atleast a 5%, more preferably 50%, and most preferably at least a 2-foldincrease or decrease relative to the corresponding property in the wildtype RANKL protein. For example, a change in osteoclast activity isevidenced by at least a 75% or greater decrease in osteoclastogenesisinitiated by a variant RANKL protein as compared to wild-type protein,and a change in binding affinity is evidenced by at least a 5% orgreater increase or decrease in binding affinity to wild-type RANKand/or OPG receptors.

[0064] By “antagonists of wild type RANKL” or grammatical equivalentsthereof herein is meant variants of RANKL that inhibit or significantlydecrease the activation of RANK receptor signaling by wild-type RANKLproteins. Both dominant negative RANKL variants and competitiveinhibitors of RANKL are antagonists of wild type RANKL. Furthermore,antagonists of wild type RANKL should not be able to appreciablyactivate the RANK receptor and initiate the RANKL signaling pathway(s).In a preferred embodiment, at least a 50% decrease in receptoractivation relative to wild type RANKL is seen, with greater than 50%,76%, 80-90% being preferred.

[0065] By “competitive inhibitor RANKL variants” or “ciRANKL” orgrammatical equivalents thereof herein is meant variants that competewith naturally occurring RANKL protein for binding to the RANK receptor,thereby limiting the ability of naturally occurring RANKL to bind andactivate the RANK receptor. In general, ci RANKL proteins are includedwithin the definition of variant RANKL proteins.

[0066] By “conformer” herein is meant a protein that has a proteinbackbone three-dimensional structure that is virtually the same as areference protein but that has significant differences in the amino acidsequence.

[0067] By “control sequences” herein is meant nucleic acid sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. Control sequences include, but are not limitedto, promoters, enhancers, and ribosome-binding site

[0068] By “epitope” herein is meant a portion of a protein that mediatesan immune response. An epitope may serve as a binding site for anantibody, T-cell receptor, and/or MHC molecule.

[0069] By “exposed residues” as used herein is meant those residueswhose side chains are significantly exposed to solvent. In a preferredembodiment, at least 30 Å² of solvent exposed area is present, withgreater than 50 Å² or 75 Å² being especially preferred. In an alternateembodiment, at least 50% of the surface area of the side chain isexposed to solvent, with greater than 75% or 90% being preferred.

[0070] By “gene therapy” herein is meant the one time or repeatedadministration of a therapeutically effective DNA, mRNA, or othernucleic acid. In one embodiment, genes are introduced into cells inorder to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene.Antisense RNA and DNA can be used as therapeutic agents for blocking theexpression of certain genes in vivo. In alternate embodiments, doublestranded RNA derived from a target gene can block the expression of atarget gene in vivo, and ribozymes can be used to process or degrade atarget gene of interest. Antisense nucleic acids can be designed tostructural genes or regulatory regions thereof.

[0071] By “hydrophobic residues” or “nonpolar residues” as used hereinis meant valine, leucine, isoleucine, methionine, phenylalanine,tyrosine, and tryptophan.

[0072] By “increase in polar character” as used herein is meant any ofthe following: (1) replacement of hydrophobic residues with neutralpolar or charged residues or (2) the replacement of neutral polarresidues with charged residues.

[0073] By “labeled” herein is meant that a protein has at least oneelement, isotope or chemical compound attached to enable the detectionand/or purification of the protein. In general, labels include, but arenot limited to, a) isotopic labels, which may be radioactive or heavyisotopes; b) immune labels, which may be antibodies or antigens; c)colored or fluorescent dyes,) enzymes, e) particles such as colloids,magnetic particles, etc.

[0074] By “linker”, “linker sequence”, “spacer”, “tethering sequence” orgrammatical equivalents thereof, herein is meant a molecule or group ofmolecules (such as a monomer or polymer) that connects two molecules andoften serves to place the two molecules in a preferred configuration. Inone aspect of this embodiment, the linker is a peptide bond. Choosing asuitable linker for a specific case where two polypeptide chains are tobe connected depends on various parameters, e.g., the nature of the twopolypeptide chains (e.g., whether they naturally oligomerize (e.g., forma dimer or not), the distance between the N- and the C-termini to beconnected if known from three-dimensional structure determination,and/or the stability of the linker towards proteolysis and oxidation.Furthermore, the linker may contain amino acid residues that provideflexibility. Thus, the linker peptide may predominantly include thefollowing amino acid residues: Gly, Ser, Ala, or Thr. These linked RANKLproteins have constrained hydrodynamic properties, that is, they formconstitutive dimers) and thus efficiently interact with other naturallyoccurring RANKL proteins to form a dominant negative heterotrimer.

[0075] The linker peptide should have a length that is adequate to linktwo RANKL variant monomers in such a way that they assume the correctconformation relative to one another so that they retain the desiredactivity as antagonists of the native RANKL protein. Suitable lengthsfor this purpose include at least one and not more than 30 amino acidresidues. Preferably, the linker is from about 1 to 30 amino acids inlength, with linkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 19 and 20 amino acids in length being preferred. See alsoWO 01/25277, incorporated herein by reference in its entirety.

[0076] In addition, the amino acid residues selected for inclusion inthe linker peptide should exhibit properties that do not interferesignificantly with the activity of the polypeptide. Thus, the linkerpeptide on the whole should not exhibit a charge that would beinconsistent with the activity of the polypeptide, or interfere withinternal folding, or form bonds or other interactions with amino acidresidues in one or more of the monomers that would seriously impede thebinding of receptor monomer domains.

[0077] Useful linkers include glycine-serine polymers (including, forexample, (GS)n, (GSGGS)n (GGGGS)n and (GGGS)n, where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers such as the tether for the shaker potassiumchannel, and a large variety of other flexible linkers, as will beappreciated by those in the art. Glycine-serine polymers are preferredsince both of these amino acids are relatively unstructured, andtherefore may be able to serve as a neutral tether between components.Secondly, serine is hydrophilic and therefore able to solubilize whatcould be a globular glycine chain. Third, similar chains have been shownto be effective in joining subunits of recombinant proteins such assingle chain antibodies.

[0078] Suitable linkers may also be identified by screening databases ofknown three-dimensional structures for naturally occurring motifs thatcan bridge the gap between two polypeptide chains. Another way ofobtaining a suitable linker is by optimizing a simple linker, e.g.,(Gly4Ser)n, through random mutagenesis. Alternatively, once a suitablepolypeptide linker is defined, additional linker polypeptides can becreated by application of PDA™ technology to select amino acids thatmore optimally interact with the domains being linked. Other types oflinkers that may be used in the present invention include artificialpolypeptide linkers and inteins. In another preferred embodiment,disulfide bonds are designed to link the two receptor monomers atinter-monomer contact sites. In one aspect of this embodiment the tworeceptors are linked at distances <5 Angstroms. In addition, the variantRANKL polypeptides of the invention may be further fused to otherproteins, if desired, for example to increase expression or stabilizethe protein.

[0079] By “mixed trimers” (frequently used interchangeably with mixedoligomers herein) is meant trimers that are composed of one or twomonomers of wild type RANKL and one or two monomers of variant RANKLprotein.

[0080] By “nonconservative modification” herein is meant a modificationin which the wild type residue and the mutant residue differsignificantly in one or more physical properties, includinghydrophobicity, charge, size, and shape. For example, modifications froma polar residue to a nonpolar residue or vice-versa, modifications frompositively charged residues to negatively charged residues or viceversa, and modifications from large residues to small residues or viceversa are nonconservative modifications.

[0081] Conservative modifications are generally those shown below,however, as is known in the art, other substitutions may be consideredconservative: Ala Ser Arg Lys Asn Gin, His Asp Glu Cys Ser Gln Asn GluAsp Gly Pro His Asn, Gln lle Leu, Val Leu lle, Val Lys Arg, Gln, Glu MetLeu, lle Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val lle,Leu

[0082] Modifications of the proteins are preferably substitutions andmay include those to surface, boundary and core areas of a TNFSF member.See, for example, U.S. Pat. Nos. 6,188,965 and 6,269,312, herebyincorporated by reference. In another preferred embodiment,modifications may be made to surface residues, particularly whenalterations to binding properties are desired (either to other monomersor to the receptor).

[0083] By “nucleic acid” herein is meant DNA, RNA, and relatedmolecules, which contain deoxy- and/or ribonucleotides. In some cases,for example for use with antisense nucleic acids, nucleic acid analogsmay be used.

[0084] By “operably linked” herein is meant that a nucleic acid isplaced into a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation.

[0085] By “patient” herein is meant both humans and other animals,particularly mammals as outlined herein, and non-animal organisms, withhumans being preferred.

[0086] By “pharmaceutically acceptable salt” as used herein refers tothose salts that are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like, or derived from inorganic bases such assodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like, as well as salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

[0087] By “polar residues” herein is meant serine, threonine, histidine,aspartic acid, asparagine, glutamic acid, glutamine, arginine, andlysine.

[0088] By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures, i.e.,“analogs” such as peptoids [see Simon et al., Proc. Natl. Acd. Sci.U.S.A. 89(20:9367-71 (1992)], generally depending on the method ofsynthesis. For example, homo-phenylalanine, citrulline, and noreleucineare considered amino acids for the purposes of the invention. “Aminoacid” also includes amino acid residues such as proline andhydroxyproline. Both D- and L-amino acids may be utilized.

[0089] By “RANKL non-agonists” or grammatical equivalents thereof hereinis meant variants of RANKL that do not appreciably activate RANKreceptor. In a preferred embodiment, activation of the RANK receptor bynon-agonistic RANKL variants is at most 50% of the activation that wouldbe attained with an equal amount of wild type RANKL protein, with atleast less than 20% or 10% being especially preferred.

[0090] By “RANKL related disorder” or “RANKL responsive disorder” orgrammatical equivalents thereof herein is meant a disorder that may beameliorated, prevented, or treated by the administration of apharmaceutical composition comprising a variant RANKL protein.RANKL-related diseases and disorders include but are not limited to:osteoporosis (postmenopausal) caused by elevated RANKL throughoophorectomy; osteoporosis (glucocorticoid-induced) caused by elevatedRANKL through glucocorticoids; rheumatoid arthritis associated withelevated RANKL in T-cells, synovial fibroblasts, and bone marrow stroma;bone loss in hyperparathyroidism caused by elevated RANKL throughparathyroid hormone (PTH); Paget's disease (sporadic) caused by elevatedRANKL; osteoclastoma associated with elevated RANKL; multiple myelomaassociated with elevated RANKL; breast cancer associated with elevatedRANKL; disuse osteopenia; bone loss due to weightlessness, malnutrition,periodontal disease, alcohol use, androgens, estrogens, chemotherapy,and parathyroid hormone; Gaucher's disease, Langerhans' cellhistiocytosis, spinal cord injury, acute septic arthritis, osteomalacia,Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrousdysplasia, periodontal reconstruction, and bone fractures; multiplemyeloma; osteolytic bone cancers such as breast cancer, lung cancer,kidney cancer and rectal cancer; bone metastasis, bone pain management,and humoral malignant hypercalcemia, among others. Additional RANKLresponsive disorders, include but are not limited to prosthesis fitting,prevention of prosthesis loosening, craniofacial reconstruction and inthe treatment of other fractures, for example, hip, spine and long bonefractures among others. Additional RANKL responsive disorders, includebut are not limited to autoimmune diseases such as systemic lupuserythematosus, inflammatory bowel disease, diabetes, multiple sclerosis,rheumatoid arthritis, and ankylosing spondylitis; as well astransplantation rejection, viral infections, and cancer. Other diseaseindications, which the variant RANKL proteins of the present inventionmay treat include, but are not limited to: hematologic neoplasisas andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, Langerhans cellhistocytosis, myeloid neoplasias such as acute myelogenous leukemias,including AML with maturation, AML without differentiation, acutepromyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia.

[0091] Additional diseases or disorders which may be treated by thevariant RANKL proteins of the present invention, include but are notlimited to cancers which rarely or never metastasize to bone and inwhich hypercalcemia generally does not occur, for example: tumors of thecentral nervous system, e.g., brain tumors (glioma, neuroblastoma,astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solidtumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer,cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine,vaginal or cervical cancers, ovarian cancer, primary liver cancer orendometrial cancer, and tumors of the vascular system (angiosarcoma andhemagiopericytoma), among others.

[0092] By “reduction in hydrophobicity” as used herein is meant theremoval of hydrophobic chemical groups.

[0093] By “soluble expression” or grammatical equivalents thereof asused herein is meant that the RANKL variant protein is expressed insoluble form (as opposed to forming aggregates or inclusion bodies). Ina preferred embodiment, greater than 5% of the expressed RANKL variantprotein is soluble, with at least 50%, 75% or 90% being especiallypreferred. In an alternate embodiment, the total yield of solubleprotein is at least 0.01 mg/L of culture, or preferably at least 0.1 or1.0 mg/L of culture. In a preferred embodiment, RANKL proteins thatexpress solubly can be released, in soluble form, from cells using anynon-denaturing buffer (that is, buffers that contain less than 1 Mguanidinium or urea).

[0094] By “treatment” herein is meant to include therapeutic treatment,as well as prophylactic, or suppressive measures for the disease ordisorder. Thus, for example, successful administration of a variantRANKL protein prior to onset of the disease may result in treatment ofthe disease. As another example, successful administration of a variantRANKL protein after clinical manifestation of the disease to combat thesymptoms of the disease comprises “treatment” of the disease.“Treatment” also encompasses administration of a variant RANKL proteinafter the appearance of the disease in order to eradicate the disease.Successful administration of an agent after onset and after clinicalsymptoms have developed, with possible abatement of clinical symptomsand perhaps amelioration of the disease, further comprises “treatment”of the disease.

[0095] By “wild-type” herein is meant an amino acid sequence or anucleotide sequence that is found in nature and includes allelicvariations; that is, an amino acid sequence or a nucleotide sequencethat has not been intentionally modified. In a preferred embodiment, thewild-type sequence is the most prevalent human sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0096] The present invention is directed to variant RANKL monomers andmixed oligomers containing the variant RANKL monomers that modifyreceptor activation. In preferred embodiments, the mixed oligomers haveeither antagonist or agonist activity. The therapeutic strategy is basedon the design of novel RANKL variants that have altered receptor bindingand/or activation properties as compared to naturally occurring RANKLproteins, and the ability to oligomerize with naturally occurring RANKLproteins. In other words, RANKL variants that result in reducedactivation (preferably do not substantially activate) RANKL receptors(as compared to a naturally occurring RANKL protein) will exchange withat least one naturally occurring RANKL protein and sequester it intoreduced or inactive hetero-oligomers, inhibiting the oligomer'sbiological activity, e.g. the ability to bind and/or activate thereceptor. Similarly, agonists can be done in a similar manner.

[0097] The RANKL variants of the present invention may be designed bymodifying RANKL proteins at key receptor contact points in order tomodify (disrupt or enhance, depending on the desired outcome) theability of the ligand to either bind or activate the receptor. Theexchange and physical interaction of these oligomeric RANKL variantswith naturally occurring RANKL proteins results in altered activity ofthe naturally occurring RANKL oligomeric proteins. To help accomplishthis goal more effectively; the RANKL variants can be designed topreferentially hetero-oligomerize with naturally occurring RANKLproteins. Alternatively, the variants may be designed to bind each otherand “swamp” out the effect of any naturally occurring RANKL proteins dueto the amount of variant oligomers present; e.g. equilibria favors thebinding of the variant mixed oligomers.

[0098] Accordingly, the present invention provides methods andcompositions utilizing variants of an extracellular domain of a RANKLprotein.

[0099] One aspect of the present invention is the generation of variantsof human RANKL that express as soluble components from host systems,preferably bacteria and mammalian host cells such as CHO cells. Afurther aspect of the present invention is the generation of variants ofhuman RANKL that act as antagonists of the wild type RANKL and/or RANKor variants of human RANKL that act as superagonists of RANK signaling.

[0100] Rational design of novel RANKL variants may be achieved by using,for example, Protein Design Automation® (PDA™) technology. (See U.S.Pat. Nos. 6,188,965; 6,269,312; 6,403,312; WO 98/47089 and U.S. Ser.Nos. 09/058,459, 09/127,926, 60/104,612, 60/158,700, 09/419,351,60/181,630, 60/186,904, 09/419,351, 09/782,004 and 09/927,790,60/347,772, and 10/218,102; and PCT/US01/218,102 and U.S. Ser. No.10/218,102, U.S. Ser. No. 60/345,805; U.S. Ser. No. 60/373,453 and U.S.Ser. No. 60/374,035, all references expressly incorporated herein intheir entirety.)

[0101] PDA™ technology couples computational design algorithms thatgenerate quality sequence diversity with experimental high-throughputscreening to discover proteins with improved properties. Thecomputational component uses atomic level scoring functions, side chainrotamer sampling, and advanced optimization methods to accuratelycapture the relationships between protein sequence, structure, andfunction. Calculations begin with the three-dimensional structure of theprotein and a strategy to optimize one or more properties of theprotein. PDA™ technology then explores the sequence space comprising allpertinent amino acids (including unnatural amino acids, if desired) atthe positions targeted for design. This is accomplished by samplingconformational states of allowed amino acids and scoring them using aparameterized and experimentally validated function that describes thephysical and chemical forces governing protein structure. Powerfulcombinatorial search algorithms are then used to search through theinitial sequence space, which may constitute 10⁵⁰ sequences or more, andquickly return a tractable number of sequences that are predicted tosatisfy the design criteria. Useful modes of the technology span fromcombinatorial sequence design to prioritized selection of optimal singlesite substitutions. PDA™ technology has been applied to numerous systemsincluding important pharmaceutical and industrial proteins and has ademonstrated record of success in protein optimization

[0102] PDA™ utilizes three-dimensional structural information. In themost preferred embodiment, the structure of RANKL is obtained by solvingits crystal structure or NMR structure by techniques well known in theart. In an alternate preferred embodiment, a homology model of RANKL isbuilt, using methods known to those in the art. For example, a homologymodel of RANKL can be made using the structure of murine RANKL and thesequence of human RANKL.

[0103] Soluble RANKL Variants

[0104] In a preferred embodiment, PDA™ technology is utilized for therational design of RANKL variants that are expressed as solubleproteins, preferably secreted, in host cell systems includingprokaryotes and eukaryotes, with preferred embodiments expressing inbacterial host cell systems. Since attempts at soluble bacterialexpression of human proteins are often unsuccessful, these solublevariants and the approaches developed to generate them have broadapplicability for the field of protein engineering. The methodsdescribed herein may be used to generate soluble variants of additionalproteins. Furthermore, the generation of soluble RANKL variants enablesthe development of RANKL variants with novel biological properties,including but not limited to RANKL variants that act as RANKLantagonists.

[0105] A variety of strategies may be utilized to design RANKL variantsthat express solubly in E. coli. In a preferred embodiment, threestrategies are used: 1) reduce hydrophobicity by replacingsolvent-exposed hydrophobic residues with suitable polar residues, 2)increase polar character by replacing neutral polar residues withcharged polar residues 3) replace non-disulfide bonded cysteine residues(unpaired cysteines) with suitable non-cysteine residues, and 4) replaceresidues whose identity is different in murine versus human RANKL. Aswill be appreciated by those in the art, several alternative strategiescould also be utilized. For example, modifications that increase thestability of a protein can sometimes improve solubility by decreasingthe population of partially folded or misfolded states. As anotherexample, protein solubility is typically at a minimum when theisoelectric point of the protein is equal to the pH of the surroundingsolution. Modifications which perturb the isoelectric point of theprotein away from the pH of a relevant environment, such as serum, cantherefore serve to improve solubility.

[0106] Replacing Solvent-Exposed Hydrophobic Residues with SuitablePolar Residues

[0107] In a preferred embodiment, solvent exposed hydrophobic residuesare replaced with structurally and functionally compatible polarresidues. Alanine and glycine may also serve as suitable replacements,constituting a reduction in hydrophobicity. Solvent exposed hydrophobicresidues can be defined according to absolute or fractional solventaccessibility, as defined above. It is also possible to use othermethods, such as contact models, to identify exposed residues. As usedherein, solvent exposed hydrophobic residues in RANKL include, but arenot limited to, isoleucine 247 and isoleucine 249.

[0108] Especially preferred solvent exposed hydrophobic residues arethose residues that have not been implicated in mediating RANKLfunction, including isoleucine 247.

[0109] In an alternate embodiment, preferred polar residues includethose that are observed at homologous positions in proteins that areRANKL homologs. In a most preferred embodiment, the RANKL homologscomprise mouse RANKL. Additional preferred RANKL homologs includeallelic variants of the human RANKL and RANKL other related species.Alternatively, RANKL homologs may comprise other TNF superfamily membersincluding but not limited to TNF-alpha, LT, TRAIL, BAFF, and CD40L. Seealso the patent application entitled “DOMINANT NEGATIVE PROTEINS ANDMETHODS THEREOF, filed on Jan. 6, 2003, Desjarlais, et al.; herebyincorporated by reference in its entirety.”

[0110] In an especially preferred embodiment, suitable polar residuesinclude only the subset of polar residues with low or favorable energiesas determined using PDA™ technology calculations. For example, suitablepolar residues may be defined as those polar residues whose energy inthe optimal rotameric configuration is more favorable than the energy ofthe exposed hydrophobic residue observed in the wild type protein atthat position, or those polar residues whose energy in the optimalrotameric configuration is among the most favorable of the set ofenergies of all polar residues at that position.

[0111] Preferred polar residues for position 247 include, but are notlimited to, glutamine, glutamic acid, lysine, and arginine.

[0112] In the most preferred embodiment, suitable polar residues includethe subset of polar residues that are deemed suitable by both PDA™calculations and by sequence alignment data.

[0113] Replacing Residues Whose Identities are Different in MurineVersus Human RANKL

[0114] In a preferred embodiment, residues whose identities vary betweenthe soluble murine ECD RANKL protein and the insoluble human ECD RANKLprotein are mutated to a structurally and functionally compatibleresidue. Variants derived from the differences between the human andmurine RANKL sequences include, but are not limited to, T173A, D174S,S183T, F201L, I207R, D230S, L231V, A232P, E234D, T243V, T254N, Y263N,S285A, E292Q, and R314Q where the human RANKL amino acid is listed firstfollowed by the murine RANKL amino acid.

[0115] In an especially preferred embodiment, positions that arehydrophobic in the human sequence and polar in the murine sequenceand/or positions that are neutral in the human sequence and charged inthe murine sequence are targeted for modification. For example,positions including but not limited to 207 and 263 may be targeted formodification. Position 207 is isoleucine, a nonpolar residue, in thehuman sequence and arginine, a charged residue, in the mouse sequence,while position 263 is tyrosine, a nonpolar residue, in the humansequence and asparagine, a polar residue, in the mouse sequence.

[0116] In a preferred embodiment, suitable residues for the above listedpositions are defined as those with low (favorable) energies ascalculated using PDA™ technology. For example, suitable residues may bedefined as those residues whose energy in the optimal rotamericconfiguration is more favorable than the energy of the wild type humanresidue observed at that position, or those residues whose energy in theoptimal rotameric configuration is among the most favorable of the setof energies of all residues at that position.

[0117] Preferred residues for position 173 include, but are not limitedto, glutamic acid and arginine.

[0118] Preferred residues for position 183 include, but are not limitedto, arginine.

[0119] Preferred residues for position 207 include, but are not limitedto, aspartic acid, glutamic acid, lysine, and arginine.

[0120] Replacing Non-Disulfide Bonded Cysteine Residues with SuitableNon-Cysteine Residues

[0121] In another preferred embodiment, free cysteine residues (that is,cysteine residues that are not participating in disulfide bonds) aremutated to a structurally and functionally compatible non-cysteineresidue. Unpaired cysteines can be identified by visual analysis of thestructure or by analysis of the disulfide bond patterns of relatedproteins. RANKL contains an unpaired cysteine at position 221.

[0122] In a preferred embodiment, if the cysteine position issubstantially buried in the protein core, suitable non-cysteine residuesinclude alanine and the hydrophobic residues, and if the cysteineposition is substantially exposed to solvent, suitable non-cysteineresidues include alanine and the polar residues.

[0123] In a preferred embodiment, suitable residues are defined as thosewith low (favorable) energies as calculated using PDA™ technology. Forexample, suitable residues may be defined as those non-cysteine residueswhose energy in the optimal rotameric configuration is more favorablethan the energy of the cysteine residue observed in the wild typeprotein at that position, or those residues whose energy in the optimalrotameric configuration is among the most favorable of the set ofenergies of all residues at that position.

[0124] In a preferred embodiment, suitable residues are defined as thosethat are observed at homologous positions in proteins that are RANKLhomologs. For example, serine is observed at position 221 in RANKLhomologs.

[0125] In a more preferred embodiment, suitable residues are those,which have both low (favorable) energies as calculated using PDA™technology and are observed at homologous positions in proteins that areRANKL homologs.

[0126] In a most preferred embodiment, cysteine 221 in RANKL is replacedby serine.

Combining Approaches

[0127] In the most preferred embodiment, multiple modifications arecombined to yield RANKL variants that express solubly in E. coli.Especially preferred combinations of modifications include, but are notlimited to, I247Q/C221 S, I247E/C221S, I247K/C221S, and I247R/C221 S.

[0128] Dominant Negative RANKL Variants

[0129] In a preferred embodiment, RANKL variants are engineered to yieldsignificantly reduced affinity and/or signaling for RANK receptorrelative to wild type RANKL while maintaining affinity for other RANKLproteins to allow formation of mixed trimers. Such RANKL variants arereferred to as “dominant negative RANKL variants” or “DN-RANKL”. Thedominant negative RANKL variants act by sequestering the naturallyoccurring RANKL proteins in mixed heterotrimers that are incapable ofappreciably activating the RANK receptor. Consequently, DN-RANKL act toantagonize the action of naturally occurring RANKL. Alternatively, theamount of variant homotrimers “swamps” out the effect of endogeneoushomotrimers.

[0130] In a preferred embodiment, DN-RANKL variant proteins exhibitdecreased biological activity as compared to wild-type RANKL, includingbut not limited to, decreased binding to the either RANK and/or OPGreceptors, decreased activation and/or a loss of osteoclastogenesisand/or costimulatory activity.

[0131] In an alternate preferred embodiment, DN-RNAKL variant proteinsdo not bind to either RANK or OPG receptors.

[0132] Variant RANKL proteins that exhibit less than 50% biologicalactivity as compared to wild-type are preferred. More preferred arevariant RANKL proteins that exhibit less than 25%, even more preferredare variant proteins that exhibit less than 15%, and most preferred arevariant RANKL proteins that exhibit less than 10% of a biologicalactivity of wild-type RANKL. Suitable assays are discussed furtherbelow.

[0133] Thus, the invention provides variant RANKL proteins with alteredbinding affinities such that the DN-RANKL proteins will preferentiallyoligomerize with wild-type RANKL, but do not substantially interact withwild type RANK or OPG receptors. “Preferentially” in this case meansthat given equal amounts of variant RANKL monomers and wild-type RANKLmonomers, at least 25% of the resulting trimers are mixed trimers ofvariant and wild type RANKL, with at least about 50% being preferred,and at least about 80-90% being particularly preferred. In other words,it is preferable that the affinity of DN-RANKL variants for wild typeRANKL is greater than the affinity of a DN-RANKL variant for anotherDN-RANKL protein or than the affinity of wild type RANKL for anotherwild type RANKL protein.

[0134] For purposes of the present invention, the areas of the wild-typeor naturally occurring RANKL molecule to be modified are preferably (butnot required to be) selected from the group consisting of the LargeDomain (also known as II), Small Domain (also known as I), the DE loop,and the trimer interface. The Large Domain, the Small Domain and the DEloop are three separate receptor contact domains, each made up ofseveral non-contiguous linear segments of the protein. These domains areidentified in the RANKL protein by comparison to the receptorinteraction domains of Lymphotoxin-alpha and TRAIL, two TNF superfamilyhomologues of RANKL whose structures (1TNR and 1D0G, respectively) havebeen defined in complex with their cognate receptors usingcrystallographic methods. The trimer interface mediates physicalinteractions between RANKL monomers. Trimerization positions can beidentified directly from the crystal structure of the mouse RANKLprotein (1JTZ). In a preferred embodiment, positions from one RANKLmonomer containing atoms that are within 5 angstroms distance from aneighboring RANKL monomer are designated as trimer interface positions.Modifications may be made solely in one of these areas or in anycombination of these and other areas.

[0135] The Large Domain preferred positions to be modified include humanRANKL positions 172, 187-193, 222-228, 267-270, 297, and 300-302. Forthe Small Domain, the preferred positions to be modified are 179-183,233-241. For the DE Loop, the preferred positions to be modified include246-253, and position 284. The Trimer Interface includes positions 163,165, 167, 193, 195, 213, 215, 217, 219, 221, 235, 237, 239, 244,253-264, 268, 271-282, 300, 302, 304-305, 307, 311, and 313-314.

[0136] Modifications to Large Domain, Small Domain, or DE loop positionsare expected to have direct effects on receptor binding and/orsignaling. As will be appreciated by those in the art, additionalmodifications outside of these domains can also indirectly affectreceptor binding and/or signaling.

[0137] Modifications at the trimer interface can be engineered tooptimize the ability of RANKL variants to hetero-trimerize withwild-type RANKL proteins. In some cases, this can be accomplished with asingle substitution at one trimer interface position. In other cases,two or more substitutions at multiple trimer interface positions must becombined. For example, the substitutions W193E and H253E could be madesimultaneously in order to create an electrostatic repulsion between twovariant RANKL monomers such that the RANKL variants would prefer tointeract with a wild-type monomer.

[0138] In a preferred embodiment, substitutions, insertions, deletionsor other modifications at multiple receptor interaction and/ortrimerization domains may be combined. Such combinations are frequentlyadvantageous in that they have additive or synergistic effects ondominant-negative activity. Examples include, but are not limited to,simultaneous substitution of amino acids at the large and small domains(e.g. 225 and 237), large domain and DE loop (e.g. 226 and 249), largedomain and trimerization domain (e.g. 225 and 215), or multiplesubstitutions within a single domain. Additional examples include anyand all combinations of substitutions.

[0139] In a most preferred embodiment, modifications at receptorinteraction and/or trimerization domains are combined with RANKLsubstitutions that confer soluble E. coli expression as described above.Examples of such combinations include but are not limited to:C221S/I247E/E269T, C221S/I247E/F270T, and C221S/I247R/H225N.

[0140] While the description herein is focused on RANKL variants, aswill be appreciated by those in the art, the embodiments and definitionscan be applied to soluble trimerizing proteins additional oligomericproteins that are involved in signal transduction.

[0141] Single Chain Dominant Negative RANKL Variants

[0142] An additional embodiment includes linked dimers of the RANKLvariants (FIG. 3). The present invention relates to these single chainpolypeptides comprising multiple receptor-interaction domains that aremodified such that each domain has significantly reduced affinity forthe cognate receptor(s). Such linked domains preferably retainassociation with individual monomer domains such that they will exhibita dominant-negative phenotype, antagonizing the action of the freemonomer domains.

[0143] RANKL Proteins As Competitive Inhibitors

[0144] In an alternative embodiment, RANKL variants are engineered toyield monomers, dimers, or trimers that bind to the RANK receptor but donot appreciably activate the RANK receptor. These variants compete withnaturally occurring RANKL protein for binding to the RANK receptor,thereby limiting the ability of naturally occurring RANKL to bind andactivate the RANK receptor. Such RANKL variants are referred to as“competitive inhibitor RANKL variants” or “ciRANKL”.

[0145] In a preferred embodiment, ciRANKL comprises two variant RANKLmonomers that are covalently connected. Such a construct would blockwild type RANKL from binding two of the three subunits that form theRANK receptor. Furthermore, the affinity of a dimeric ciRANKL wouldlikely be higher than an equivalent monomeric ciRANKL, facilitatingcompetition. Linkers include, but are not limited to, polypeptidelinkages between N- and C-termini of the domains, linkage via adisulfide bond between monomers, and linkage via chemical cross-linkingreagents. Alternatively, the N- and C-termini may be covalently joinedby deletion of portions of the N- and/or C-termini and linking theremaining fragments via a linker or linking the fragments directly.

[0146] In a preferred embodiment, ciRANKL variant proteins exhibitdecreased biological activity as compared to wild-type RANKL, includingbut not limited to, decreased activation and/or a loss ofosteoclastagenesis. Suitable assays include, but are not limited to,those described below. Furthermore, ciRANKL proteins are capable ofinhibiting the biological functioning of wild type RANKL.

[0147] Variant RANKL proteins that reduce the biological activity ofwild-type RANKL by at least 50% are preferred. More preferred arevariant RANKL proteins reduce the biological activity of wild type RANKLby 75%. Especially preferred ciRANKL variants reduce the activity ofwild-type RANKL by at least 90%.

[0148] Thus, the invention provides variant RANKL proteins with alteredbinding affinities such that the ciRANKL proteins will form monomers ordimers, but not trimers, and will bind to the RANK receptor. In apreferred embodiment, the affinity of ciRANKL for the RANK receptor isgreater than the affinity of wild type RANKL for the RANK receptor. Itis especially preferred that ciRANKL binds to RANK with at least 10-foldgreater affinity than the wild type RANKL.

[0149] For purposes of the present invention, the areas of the wild-typeor naturally occurring RANKL molecule to be modified are selected fromthe group consisting of the Large Domain (also known as II), SmallDomain (also known as I), the DE loop, and the trimer interface. TheLarge Domain, the Small Domain and the DE loop are three separatereceptor contact domains, each made up of several non-contiguous linearsegments of the protein. These domains are identified in the RANKLprotein by comparison to the receptor interaction domains ofLymphotoxin-alpha and TRAIL, two TNF superfamily homologues of RANKLwhose structures (1TNR and 1D0G, respectively) have been defined incomplex with their cognate receptors using crystallographic methods. Thetrimer interface mediates physical interactions between RANKL monomers.Trimerization positions can be identified directly from the crystalstructure of the mouse RANKL protein (1JTZ). In a preferred embodiment,positions from one RANKL monomer containing atoms that are within 5angstroms distance from a neighboring RANKL monomer are designated astrimer interface positions. Modifications may be made solely in one ofthese areas or in any combination of these and other areas.

[0150] Linkers

[0151] In a preferred embodiment, a linker peptide is chosen such thatthe two RANKL variant monomers assume a conformation that allows bindingto the RANK receptor. Linkers are defined above.

[0152] RANKL Superagonist Variants

[0153] In a preferred embodiment, PDA™ technology is utilized for therational design of RANKL variants that behave as superagonists. RANKLsuperagonist variants are RANKL variants that bind to and/or activatethe RANK receptor more strongly than the wild type human RANKL does. Asa result, RANKL superagonists could be used as a treatment forRANKL-responsive conditions that result from insufficient activity ofendogenous RANKL.

[0154] In a preferred embodiment, RANKL superagonists exhibit increasedbiological activity as compared to wild type RANKL, including but notlimited to increased binding to RANK receptor, increased activation ofthe RANK receptor, and/or increase in cytotoxic activity. In a preferredembodiment the RANKL superagonist proteins are at least 10% more activethen wild type human RANKL, with at least 50%, 100% or 200% increases inactivity being especially preferred. Suitable assays are discussedfurther below.

[0155] For purposes of the present invention, the areas of the wild typeor naturally occurring RANKL molecule to be modified are selected fromthe group consisting of the Large Domain (also known as II), SmallDomain (also known as I), the DE loop, and the trimer interface. TheLarge Domain, the Small Domain and the DE loop are three separatereceptor contact domains, each made up of several non-contiguous linearsegments of the protein. These domains are identified in the RANKLprotein by comparison to the receptor interaction domains ofLymphotoxin-alpha and TRAIL, two TNF superfamily homologues of RANKLwhose structures (1TNR and 1D0G, respectively) have been defined incomplex with their cognate receptors using crystallographic methods. Thetrimer interface mediates interactions between RANKL monomers.Trimerization positions can be identified directly from the crystalstructure of the mouse RANKL protein (1JTZ). In a preferred embodiment,positions from one RANKL monomer containing atoms that are within 5angstroms distance from a neighboring RANKL monomer are designated astrimer interface positions. Modifications may be made solely in one ofthese areas or in any combination of these and other areas.

[0156] Generation of Nucleic Acids Encoding RANKL Variants

[0157] In a preferred embodiment, nucleic acids encoding RANKL variantsare prepared by total gene synthesis, or by site-directed mutagenesis ofthe DNA encoding wild type or variant RANKL protein. Methods includingtemplate-directed ligation, recursive PCR, cassette mutagenesis,site-directed mutagenesis or other techniques that are well known in theart may be utilized.

[0158] Using the nucleic acids of the present invention, which encode avariant RANKL protein, a variety of expression vectors can be made.Preferred bacterial expression vectors include but are not limited topET, pBAD, bluescript, pUC, pQE, pGEX, pMAL, and the like. Generally,these expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleic acid encoding thevariant RANKL protein. Transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. Furthermore, the vector will typically include a selectablemarker such as an antibiotic resistance gene.

[0159] A suitable bacterial promoter is any nucleic acid sequencecapable of binding bacterial RNA polymerase and initiating thedownstream (3′) transcription of the coding sequence of the variantRANKL protein into mRNA. A bacterial promoter has a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region typically includesan RNA polymerase binding site and a transcription initiation site.Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter may include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

[0160] In addition to a functioning promoter sequence, an efficientribosome binding site is desirable. In E. coli, the ribosome bindingsite is called the Shine-Delgarno (SD) sequence and includes aninitiation codon and a sequence 3-9 nucleotides in length located 3-11nucleotides upstream of the initiation codon.

[0161] The expression vector may also include a signal peptide sequencethat provides for secretion of the variant RANKL protein in bacteria.The signal sequence typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell, as is well known in the art. The protein is either secretedinto the growth media (gram-positive bacteria) or into the periplasmicspace, located between the inner and outer membrane of the cell(gram-negative bacteria). For expression in bacteria, usually bacterialsecretory leader sequences, operably linked to a variant RANKL encodingnucleic acid, are preferred.

[0162] The bacterial expression vector may also include a selectablemarker gene to allow for the selection of bacterial strains that havebeen transformed. Suitable selection genes include genes, which renderthe bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

[0163] Suitable expression vectors for non-bacterial expression systemsare also well known in the art and can be utilized.

[0164] In an alternate embodiment, variant RANKL proteins or fragmentsthereof may be prepared by chemical synthesis. In such an embodiment, itis not necessary to generate nucleic acids encoding the RANKL variants.

[0165] Expression of RANKL Variants

[0166] In a most preferred embodiment, the variant RANKL proteins areexpressed in bacterial systems, including but not limited to E. coli.Bacterial expression systems and methods for their use are well known inthe art (see Current Protocols in Molecular Biology, Wiley & Sons, andMolecular Cloning—A Laboratory Manual—3rd Ed., Cold Spring HarborLaboratory Press, New York (2001)). The choice of codons, suitableexpression vectors and suitable host cells will vary depending on anumber of factors, and may be easily optimized as needed.

[0167] Bacterial expression vectors encoding RANKL variants aretransformed into bacterial host cells using techniques well known in theart, such as calcium chloride treatment, electroporation, and others.

[0168] In a preferred embodiment, bacterial cultures are grown tomid-log phase and expression is induced, for instance with IPTG. Cellsare then harvested after 2-24 hours. Protein can be released from thecells using several methods, including sonication, addition ofdetergents, French press, etc.

[0169] In an alternate embodiment, variant RANKL proteins are expressedin non-bacterial systems, including but not limited to yeast,baculovirus, and mammalian expression systems, as well as in vitroexpression systems. Suitable protocols are well known in the art.

[0170] Purification of RANKL Variants

[0171] In a preferred embodiment, the variant RANKL protein is purifiedor isolated after expression. Standard purification methods includeelectrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the variant RANKL protein may be purified using a standardanti-recombinant protein antibody column. Ultrafiltration anddiafiltration techniques, in conjunction with protein concentration, arealso useful. For general guidance in suitable purification techniques,see Scopes, R., Protein Purification, Springer-Verlag, NY, 3r ed (1994).The degree of purification necessary will vary depending on the use ofthe variant RANKL protein. In some instances no purification will benecessary.

[0172] Derivitization of RANKL Variants

[0173] Once made, the variant RANKL proteins may be covalently ornon-covalently modified. Derivatized RANKL variants may exhibit improvedsolubility, absorption, immunogenicity, pharmacokinetics, and the like.Moieties capable of mediating such effects are disclosed, for example,in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,Easton, Pa. (1980).

[0174] Synthetic Modification

[0175] One type of covalent modification includes reacting targetedamino acid residues of a variant RANKL polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N-or C-terminal residues of a variant RANKL polypeptide.

[0176] Examples include, but are not limited to, modification to effectbinding to human serum albumin, alkylaton of any side chain, lipidation,acetylation, acylation, nitrile derivatives of asparagine or glutamine,sulfoxide derivatives methionine, cysteinyl residues reacted withcompounds including alpha-haloacetates, histidyl residues derivatized byreaction with diethylprocarbonate, and lysinyl and amino terminalresidues reacted with compounds such as succinic or other carboxylicacid anhydrides.

[0177] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

[0178] Derivatization with bifunctional agents is useful, for instance,for cross linking a variant RANKL protein to a water-insoluble supportmatrix or surface for use in the method for purifying anti-variant RANKLantibodies or screening assays. Commonly used cross linking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithiol propioimidate.

[0179] Other modifications may be made to the variant RANKL proteins ofthe present invention, including modifications to the protein thatenhance stability, dosage administration (e.g., amphiphilic polymers,see WO 0141812A2, commercially available from Nobex Corporation),clearance (e.g., PEG, aliphatic moieties that effect binding to HSA),and the like.

[0180] Glycosylation

[0181] The sequence of RANKL variant proteins can be further modified toadd or remove glycosylation sites. For example, O-linked glycosylationsites can be altered by adding or removing one or more serine orthreonine residues. N-linked glycosylation sites can be altered byincorporating or removing a canonical N-linked glycosylation site,N-X-Y, where N is asparagine, X is any amino acid except for proline andY is threonine, serine or cysteine. Another means of increasing thenumber of carbohydrate moieties on the variant RANKL polypeptide is bychemical or enzymatic coupling of glycosides to the polypeptide. Suchmethods are described in the art, e.g., in WO 87/05330 published Sep.11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306(1981).

[0182] PEGylation

[0183] Another type of covalent modification of variant RANKL compriseslinking the variant RANKL polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. ARE ALL SHEARWATER AND ENZON CASES CITED HERE???? Thesenonproteinaceous polymers may also be used to enhance the ability of avariant RANKL to disrupt receptor binding or to alter the stability,solubility, pharmacokinetics, and/or immunogenicity of the variantRANKL.

[0184] In another preferred embodiment, the location of cysteine,lysine, and/or histidine residues in a RANKL variant is modified inorder to control the site of PEG attachment. A variety of couplingchemistries may be used to achieve PEGylation, as is well known in theart. Examples, include but are not limited to, the technologies ofShearwater and Enzon. See, Kinstler et al, Advanced Drug DeliveriesReviews, 54, 477-485 (2002) and M J Roberts et al, Advanced DrugDelivery Reviews, 54, 459-476 (2002), both hereby incorporated byreference.

[0185] Optimal sites for modification can be chosen using a variety ofcriteria, including but not limited to, visual inspection, structuralanalysis, sequence analysis and molecular simulation. For example, thefractional accessibility of individual residues can be analyzed toidentify modification sites that will not disrupt the monomer structure.In additional preferred embodiments, modification sites are chosen suchthat the distance from the modification site to another RANKL monomer ismaximal. In additional embodiments, it is possible that receptor bindingdisruption may occur and may be beneficial to the activity of the RANKLvariants of this invention.

[0186] Circular Permutation and Cyclization

[0187] In another preferred embodiment, the wild type RANKL or variantsgenerated by the invention may be circularly permuted. All naturalproteins have an amino acid sequence beginning with an N-terminus andending with a C-terminus. The N- and C-termini may be joined to create acyclized or circularly permutated RANKL proteins while retaining orimproving biological properties (e.g., such as enhanced stability andactivity) as compared to the wild-type protein. In the case of a RANKLprotein, a novel set of N- and C-termini are created at amino acidpositions normally internal to the protein's primary structure, and theoriginal N- and C-termini are joined via a peptide linker consisting offrom 0 to 30 amino acids in length (in some cases, some of the aminoacids located near the original termini are removed to accommodate thelinker design). In a preferred embodiment, the novel N- and C-terminiare located in a non-regular secondary structural element, such as aloop or turn, such that the stability and activity of the novel proteinare similar to those of the original protein. In a further preferredembodiment PDA™ technology may be used to further optimize the RANKLvariant, particularly in the regions created by circular permutation.These include the novel N- and C-termini, as well as the originaltermini and linker peptide. In addition, a completely cyclic RANKL maybe generated, wherein the protein contains no termini. This isaccomplished utilizing intein technology. Thus, peptides can be cyclizedand in particular inteins may be utilized to accomplish the cyclization.

[0188] Various techniques may be used to permutate proteins. See U.S.Pat. No. 5,981,200; Maki K, Iwakura M., Seikagaku. January 2001; 73(1):42-6; Pan T., Methods Enzymol. 2000; 317:313-30; Heinemann U, Hahn M.,Prog Biophys Mol Biol. 1995; 64(2-3): 121-43; Harris M E, Pace N R, MolBiol Rep. 1995-96; 22(2-3): 115-23; Pan T, Uhlenbeck O C., Mar 30, 1993;125(2): 111-4; Nardulli A M, Shapiro D J. 1993 Winter; 3(4): 247-55, EP1098257 A2; WO 02/22149; WO 01/51629; WO 99/51632; Hennecke, et al.,1999, J. Mol. Biol., 286, 1197-1215; Goldenberg et al J. Mol. Biol 165,407-413 (1983); Luger et al, Science, 243, 206-210 (1989); and Zhang etal., Protein Sci 5, 1290-1300 (1996); all hereby incorporated byreference.

[0189] Fusion Constructs

[0190] Variant RANKL polypeptides of the present invention may also befused to another, heterologous polypeptide or amino acid sequence toform a chimera. The chimeric molecule may comprise a fusion of a variantRANKL polypeptide with an immunoglobulin or a particular region of animmunoglobulin such as the Fc or Fab regions of an IgG molecule.). Insome embodiments, for example in the creation of animal models ofdisease, fusion proteins comprising the variant RANKL proteins withother sequences may be done, for example using fusion partnerscomprising labels (e.g. autofluorescent proteins, survival and/orselection proteins), stability and/or purification sequences, toxins,variant proteins from other members of the superfamily (e.g. analogousto the creation of “bi-specific antibodies”) or any other proteinsequences of use. Additional fusion partners are described below. Insome instances, the fusion partner is not a protein.

[0191] In another embodiment, the RANKL variant is fused with humanserum albumin to effect an improvement in pharmacokinetics.

[0192] In another preferred embodiment, the RANKL variant is conjugatedto an antibody, preferably an anti-variant RANKL protein antibody.

[0193] In a further embodiment, RANKL is fused to a cytotoxic agent. Inthis method, the RANKL fusion acts to target the cytotoxic agent totumor tissue or cells, resulting in a reduction in the number ofafflicted cells. Such an approach thereby reduces symptoms associatedwith cancer and RANKL protein related disorders. Cytotoxic agentsinclude, but are not limited to, diphtheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin andthe like, as well as radiochemicals.

[0194] Peptide Tags

[0195] Various tag polypeptides and their respective antibodies are wellknown in the art. Epitope tags may be placed at the amino-orcarboxyl-terminus of the variant RANKL proteins to enable antibodydetection. Also, the epitope tag enables the variant RANKL protein to bereadily purified by affinity purification. Examples of peptide tagsinclude, but are not limited to, poly-histidine (poly-His) orpoly-histidine-glycine (poly-His-Gly) tags; the flu HA tag polypeptide[Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag [Evanet al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; the HerpesSimplex virus glycoprotein D (gD) tag [Paborsky et al., ProteinEngineering, 3(6):547-553 (1990)], the Flag-peptide [Hopp et al.,BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide [Martin etal., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A.87:6393-6397 (1990)].

[0196] Labels

[0197] In one embodiment, the variant RANKL protein is modified by theaddition of one or more labels. For example, labels that may be used arewell known in the art and include but are not limited to biotin, tag andfluorescent labels (e.g. fluorescein). These labels may be used invarious assays as are also well known in the art to achievecharacterization.

[0198] Assays

[0199] Variant RANKL proteins may be experimentally tested and validatedusing in vivo and in vitro assays. Suitable assays include, but are notlimited to, activity assays and binding assays. For example, RANKLactivity assays, such as the tartrate resistant acid phosphatase assay(TRAP; Matsuzaki et al., Biochemical and biophysical researchcommunications 246, 199-204 (1998)) for monitoring the differentiationof pre-osteoclast or RAW264.7 cells into osteoclasts, and the NF-□B (Weiet al., Endocrinology 142, 1290-1295, (2001)) or c-Jun (Srivastava etal., JBC 276, 8836-8840 (2001)) transcription factor activation assaysfor monitoring signaling through RANK are screens that may be utilizedin identifying RANKL variants that are antagonists of wild-type RANKL.Other biological markers for osteoclastogenesis include countingmultinucleated TRAP staining cells, calcitonin receptor expression, thepresence of ruffled borders on osteoclasts, and cathepsin K expressionand activity (see Suda et al., Modulation of Osteoclast Differentiationand Function by the New Members of the Tumor Necrosis Factor Receptorand Ligand Families; Endocrine Reviews 20(3):345-357 (1999) and Garneroet al., The collagenolytic activity of cathepsin K is unique amongmammalian proteinases; Journal of biochemistry 273(48):32347-32352(1998)).

[0200] In a preferred embodiment, binding affinities for the followinginteractions are determined and compared: 1) variant RANKL oligomerformation, 2) wild type RANKL oligomer formation, 3) variant RANKLbinding to RANK, 4) wild type RANKL binding to RANK, 5) variant RANKLbinding to OPG, and 6) wild type RANKL binding to OPG. Suitable assaysinclude, but are not limited to, quantitative comparisons comparingkinetic and equilibrium binding constants. The kinetic association rate(Kon) and dissociation rate (Koff), and the equilibrium bindingconstants (Kd) may be determined using surface plasmon resonance on aBIAcore instrument following the standard procedure in the literature[Pearce et al., Biochemistry 38:81-89 (1999)]. Several alternativemethods can also be used to determine binding affinity and kinetics.

[0201] RANKL variants can also be tested to determine whether they arecapable of forming mixed oligomers including but not limited to mixedtrimers. In a preferred embodiment, this is accomplished by labelingwild type RANKL and variant RANKL with distinguishable tags, combiningwild type and variant RANKL, and screening for oligomers that containboth tag types. For example, FLAG-tagged wild type RANKL and His-taggedvariant RANKL can be combined, and sandwich ELISAs can be performed toidentify trimers that contain both FLAG and His tag. Another alternativeis to run native gels and perform Western blots using both anti-FLAG andanti-His tag antibodies. This method relies on the fact that FLAG andHis tags significantly perturb protein migration in native gels. As willbe appreciated by those in the art, many alternate protocols could alsobe used to measure the formation of mixed trimers.

[0202] Therapeutic Application of RANKL Variants

[0203] Once made, the variant RANKL proteins and nucleic acids of theinvention find use in a number of applications. In a preferredembodiment, the variant RANKL proteins are administered to a patient totreat a RANKL related disorders.

[0204] Pharmaceutical Composition

[0205] The pharmaceutical compositions of the present invention comprisea variant RANKL protein in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water-soluble form, for example as pharmaceutically acceptablesalts. Particularly preferred are the ammonium, potassium, sodium,calcium, and magnesium salts.

[0206] The pharmaceutical compositions may also include one or more ofthe following: carrier proteins such as serum albumin; buffers such asNaOAc; fillers such as microcrystalline cellulose, lactose, corn andother starches; binding agents; sweeteners and other flavoring agents;coloring agents; and polyethylene glycol. Additives are well known inthe art, and are used in a variety of formulations.

[0207] In a further embodiment, the variant RANKL proteins are added ina micellular formulation; see U.S. Pat. No. 5,833,948, hereby expresslyincorporated by reference in its entirety.

[0208] Combinations of pharmaceutical compositions may be administered.Moreover, the compositions may be administered in combination with othertherapeutics.

[0209] Depending upon the manner of introduction, the pharmaceuticalcomposition may be formulated in a variety of ways. The concentration ofthe therapeutically active variant RANKL protein in the formulation mayvary from about 0.1 to 100 weight %. In another preferred embodiment,the concentration of the variant RANKL protein is in the range of 0.003to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimolesper kilogram of body weight being preferred.

[0210] Also, sustained release or controlled release formulations may beused for the compositions of the present invention. For example,ProLease® (commercially available from Alkermes) a microsphere-baseddelivery system composed of the desired bioactive molecule incorporatedinto a matrix of poly-DL-lactide-co-glycolide (PLG) and otherpharmaceutically compatible polymeric matrices may be used to createsustained release formulations.

[0211] Route of Administration

[0212] The administration of the variant RANKL proteins of the presentinvention, preferably in the form of a sterile aqueous solution, may bedone in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx®)inhalable technology commercially available from Aradigm or Inhance™pulmonary delivery system commercially available from InhaleTherapeutics), vaginally, rectally, or intraocularly. In some instances,for example, in the treatment of wounds, inflammation, etc., the variantRANKL protein may be directly applied as a solution or spray. Dependingupon the manner of introduction, the pharmaceutical composition may beformulated in a variety of ways.

[0213] Dosing

[0214] In a preferred embodiment, a therapeutically effective dose of avariant RANKL protein is administered to a patient in need of treatment.The exact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for variant RANKL protein degradation,systemic versus localized delivery, and the rate of new proteasesynthesis, as well as the age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

[0215] Gene Therapy

[0216] Similarly, nucleic acid encoding the variant RANKL proteins(including both the full-length sequence, partial sequences, orregulatory sequences of the variant RANKL coding regions) may beadministered in gene therapy applications.

[0217] In a preferred embodiment, the nucleic acid encoding the variantRANKL proteins (including both the full-length sequence, partialsequences, or regulatory sequences of the variant RANKL coding regions)may also be used in gene therapy. In gene therapy applications, genesare introduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product, for example for replacementof a defective gene. Antisense RNA and DNA can be used as therapeuticagents for blocking the expression of certain genes in vivo. It hasalready been shown that short antisense oligonucleotides can be importedinto cells where they act as inhibitors (see Zamecnik et al., Proc.Natl. Acad. Sci. U.S.A. 83:4143-4146 (1986)). The oligonucleotides canbe modified to enhance their uptake, e.g. by substituting theirnegatively charged phosphodiester groups by uncharged groups.

[0218] There are a variety of techniques available for introducingnucleic acids into viable cells. The currently preferred in vivo genetransfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. U.S.A. 87:3410-3414 (1990). For review of gene marking andgene therapy protocols see Anderson et al., Science 256:808-813 (1992).

[0219] In a preferred embodiment, variant RANKL genes are administeredas DNA vaccines, either single genes or combinations of variant RANKLgenes. Naked DNA vaccines are generally known in the art. Brower, NatureBiotechnology, 16:1304-1305 (1998). Methods for the use of genes as DNAvaccines are well known to one of ordinary skill in the art, and includeplacing a variant RANKL gene or portion of a variant RANKL gene underthe control of a promoter for expression in a patient in need oftreatment.

[0220] The variant RANKL gene used for DNA vaccines may encodefull-length variant RANKL proteins, but more preferably encodes portionsof the variant RANKL proteins including peptides derived from thevariant RANKL protein. In a preferred embodiment a patient is immunizedwith a DNA vaccine comprising a plurality of nucleotide sequencesderived from a variant RANKL gene. Similarly, it is possible to immunizea patient with a plurality of variant RANKL genes or portions thereof asdefined herein. Without being bound by theory, expression of thepolypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced which recognize and destroy oreliminate cells expressing RANKL proteins.

[0221] In a preferred embodiment, the DNA vaccines include a geneencoding an adjuvant molecule with the DNA vaccine. Such adjuvantmolecules include cytokines that increase the immunogenic response tothe variant RANKL polypeptide encoded by the DNA vaccine. Additional oralternative adjuvants are known to those of ordinary skill in the artand find use in the invention.

[0222] All references cited herein, including patents, patentapplications (provisional, utility and PCT), and publications areincorporated by reference in their entirety.

EXAMPLES Example 1

[0223] Solubility Library Design

[0224] A homology model of human RANKL based on the mouse RANKLstructure (1JTZ) was analyzed for excessive exposure of non-polar aminoacids on the surface of the protein.

[0225] The solvent accessible surface area was determined for each aminoacid in the structure using algorithms known in the art. The mostdramatic exposure of non-polar residues was observed at I247 isoleucine247 (I247) (138 Å²) and isoleucine 249 (1249) (108 Å²). Indeed, thesetwo isoleucine residues are the only non-polar amino acids withaccessible surface area exceeding 100 Å² in the structural context oftrimerized RANKL. I249, however, is located in a region of the proteinpredicted to interact directly with RANK. Therefore, I247 was chosen forsubstitution. PDA™ technology predicted that multiple substitutions atI247 were allowed, including the charged amino acids E, K, glutamicacid, lysine and Rarginine, and the polar amino acid Qglutamine. Suchsubstitutions, due to their intrinsically polar nature, are expected toenhance the solubility of the folded protein, and in some cases assistin its proper folding after expression in E. coli. The PDA™ technologypredictions suggest also that the aforementioned substitutions willpreserve tertiary and quaternary structure of the RANKL protein, suchthat it will retain a large part of its functional activity.

[0226] Additional analysis was performed at the primary structure levelby comparing the human RANKL sequence (FIGS. 1a, 1 b) to that of themouse sequence (FIG. 1c). Although the mouse and human sequences differat only sixteen positions (FIG. 2a) within our construct, the mouseprotein expresses solubly in E. coli while the human protein does not.This analysis highlighted additional positions for substitution. Ofparticular interest are positions containing a non-polar amino acid inthe human sequence and a polar amino acid in the mouse sequence. Forinstance, isoleucine 207 in the human RANKL sequence is replaced by anarginine in the mouse sequence. This substitution was also consistentwith PDA™ technology predictions that arginine is favorable at thisposition in the structure. At several other positions (e.g. threonine173, serine 183), variability between the mouse and human sequence wasused simply as an indicator that substitution at those positions mightbe compatible with the protein structure. In these cases, PDA™technology simulations indicated that replacement with charged aminoacids is permissible.

[0227] Finally, a single unpaired C cysteine exists in the human RANKLprotein at position 221. As is well known in the art, unpaired cysteinesoften lead to unfavorable solution properties of a protein. Therefore,this residue in the human sequence was replaced by serine. A serine atthis position is predicted by PDA™ technology simulations to befavorable. In addition, inspection of a multiple sequence alignment ofthe TNF ligand superfamily reveals that S serine occurs at theequivalent position in other members of the TNF family.

[0228] A summary of the amino acid sequences for the human RANKLsolubility variants is listed in FIG. 2b.

[0229] RANKL Solubility Variants Library Construction

[0230] The full-length RANKL cDNA was cloned using RT-PCR on lymph nodecDNAs. Amino acids 159 to 317 were sub-cloned into pET33b afterPCR-mediated addition of TEV and HIS His tags. The amino acid sequenceof this wild-type RANKL is described in FIG. 1b.

[0231] Site directed mutagenesis was performed on plasmid cDNA clones tointroduce the nucleotide changes that result in the amino acidsubstitutions listed below. The site directed mutagenesis reactionutilized pairs of divergent and overlapping oligonucleotides withnucleotide variations that introduce the specific amino acid changes.These oligonucleotides were extended using Pfu polymerase throughmultiple rounds of thermal cycling. The template plasmid was degradedusing the restriction enzyme DpnI, and the extension products weretransformed into TOP10 bacterial cells. Clones were sequenced to confirmthe presence of the designed changes and to insure no unwantedmodifications were introduced.

[0232] RANKL Solubility Variant Bacterial Expression

[0233] The human RANKL variants were tested for soluble expression inbacteria (FIGS. 4-8). The sequence confirmed plasmids were transformedinto BL21(DE3)pLysS E. coli cells. After bacterial growth, inductionwith IPTG, and expression, the cells were lysed and centrifuged toseparate the insoluble and soluble fraction of the lysate. SDS-PAGE andWestern blot analysis were performed to characterize expression of theRANKL solubility variants. The results of the expression analysis aresummarized in Table 1 below. The results show that the approach togenerate soluble human RANKL was successful, as ten variants showsoluble expression in bacterial. TABLE 1 Summary of the bacterialexpression analysis using human RANKL solubility variants. RANKL VariantBacterial Expression Wild-type RANKL No Soluble Expression(Nter-HIS-Tev-159-317) C221S No Soluble Expression I247E No SolubleExpression I247K No Soluble Expression I247Q No Soluble ExpressionT173E/C221S No Soluble Expression T173R/C221S No Soluble ExpressionS183R/C221S No Soluble Expression I207D/C221S No Soluble ExpressionI207E/C221S No Soluble Expression I207K/C221S Soluble ExpressionI207R/C221S Low Soluble Expression C221S/A232K Very Low SolubleExpression C221S/I247A Soluble Expression C221S/I247D Soluble ExpressionC221S/I247E Soluble Expression C221S/I247K Soluble ExpressionC221S/I247Q Soluble Expression C2213/I247R Low Soluble ExpressionT173R/S183R/I207R/C221S/I247R Low Soluble Expression

[0234] The significance of the C221S change in imparting solubility tothe variants was examined by creating variants with the single pointmodifications C221S, I247Q, I247E, and I247K. Expression analysis showedthat soluble expression was observed when C221 S was combined with oneof the three changes, I247Q, I247E, and I247K (FIGS. 7, 8).

[0235] RANKL Solubility Variant Characterization

[0236] Six of the highest expressing RANKL solubility variantsC221S/I207K, C221S/I247A, C221S/I247D, C221S/I247E, C221S/I247K, andC221S/I247Q were scaled up for protein purification (FIGS. 9, 10) andbiochemical characterization. Highly purified protein was collected fromthe soluble fractions. The proteins were expressed with good yields anddetermined to be trimeric using dynamic light scattering and sizeexclusion chromatography (FIG. 11, 12). In addition, the six RANKLsolubility variants were shown to bind RANK receptor and the decoyreceptor OPG in immunoprecipitation experiments (FIG. 13, 14). Thesolubility variants express well, can be purified effectively, fold andtrimerize as expected, and bind the RANK receptor and OPG receptors.

[0237] The human RANKL solubility variants were evaluated for theirability to activate the NFκB and JNK pathways in the mouse macrophagemonocytic cell line RAW 264.7. RANKL binding to the RANK receptor isknown to activate these two pathways (see Gowen, M., Every, J G, andKumar S. Emerging therapies for osteoporosis. Emerging Drugs, 2000 5(1):p.1-43; Rodan, G A, and Martin T J. Therapeutic approaches to bonedisease. Science 2000 289: p. 1508-1514; Hofbauer, L C, Neubauer, A, andHeufelder A E. Receptor activator of nuclear factor-kB ligand andosteoprotegrin. 2001 Cancer 92(3): p.460-470.). Activating thesepathways requires a RANKL protein that is folded, trimeric, and capableof binding the RANK receptor. The variant C221S/I247E activates bothNFκB and JNK (FIGS. 15, 16) to similar levels as does the wild-typemouse RANKL (commercially available from R&D Systems).

[0238] The six human RANKL solubility variants were evaluated for theirability to activate osteoclastogenesis in the mouse monocytic cell lineRAW 264.7. The RAW 264.7 cells are known to differentiate intomulti-nucleated osteoclasts in a RANKL dependent manner (see Hitoshi H.,Sakai E., Kanaoka K., Saito K., Matsuo K. I., Kitaura H., Yoshida N.,and Koji Nakayama. U0126 and PD98059, specific inhibitors of MEK,accelerate differentiation of RAW264.7 cells into osteoclast-like cells.J. Biol. Chem, 10.1074/jbc.M208284200). Osteoclast formation can bemonitored by the production of Tartrate Resistant Acid Phosphatase(TRAP) from the RAW 264.7 cells as they differentiate. RAW264.7 cellstreated with increasing doses of RANKL variant C221S/I247E reveal adose-dependent increase in the number of multi nucleated cells stainingfor TRAP (FIG. 17). A quantitative TRAP assay using p-nitrophenylphosphate as substrate (see Nakagawa N, Kinosaki M, Yamaguchi K, ShimaN, Yasuda H, Yano K, Morinaga T, Higashio K. RANK is the essentialsignaling receptor for osteoclast differentiation factor inosteoclastogenesis. Biochem Biophys Res Commun Dec. 18, 1998;253(2):395-400.) shows that this variant also mediatesosteoclastogenesis with an activity similar to commercially availableRANKL made in E. coli and refolded from occlusion bodies (commerciallyavailable from BioSource) (FIG. 18). All six solubility variantsdemonstrate similar osteoclastogenesis activity using the TRAP assay(FIG. 19). The ability to antagonize the RANKL solubility variantC221S/I247E with soluble receptor RANK-Fc and OPG-Fc (FIG. 20) confirmsthat the variant binds RANK and OPG and that the osteoclastogenesisobserved in the TRAP assay results from the binding of RANKL to the RANKreceptor.

[0239] Tartrate-Resistant Acid Phosphatase (TRAP) Activity Bioassay

[0240] RAW 264.7 cells (ATCC#TIB-71) were cultured and maintained in 75cm² flasks in 5% CO₂ humidified incubator at 37° C. in growth mediacontaining α-MEM w/2 mM L-Glutamine (Gibco-BRL#12571-063), 10% HeatInactivated Fetal Bovine Serum (FBS) (Hyclone#SH30071.03) andPenicillin/Streptomycin (Gibco-BRL#15140-122) 100 units/ml and 100 μg/mlrespectively.

[0241] Recombinant mouse RANKL (R&D#462-TR), recombinant human solubleRANKL (Biosource#PHP0034), and recombinant human soluble RANKL variantswere diluted to their working concentration in assay media consisting ofα-MEM w/I-glutamine (Gibco-BRL#12571-063) supplemented with 10% HeatInactivated Fetal Bovine Serum (FBS) (Hyclone#SH30071.03) andPenicillin/Streptomycin (Gibco-BRL#15140-122) 100 units/ml and 100 μg/mlrespectively. 250 μl of RANKL standards and samples were added to96-well assay plate in triplicate or quadruplicate, and serially diluted1:2 by transferring 125 μl into subsequent wells containing 125 μl ofassay media.

[0242] Aspirating the exhausted media, adding 10 ml of pre-warmed mediato the flask, and scraping cells from the flask using a cell scraperharvested the cells. The cells were transferred to a 15 ml conical tube,spun at 1000 rpm for 5 minutes and resuspended in 10 ml of assay media.The cells were counted and seeded at density of 1.5×10³/125 μl/well in96-well assay plate. Assay plates were incubated at 37° C. in 5% CO₂humidified incubator for 72 hours.

[0243] The amount of osteoclast-like cells induced from the RAW 264.7cells was determined by measuring the Tartrate-Resistant AcidPhosphatase (TRAP) activity. TRAP substrate was prepared by adding 20 mgof p-nitrophenol phosphate (pNPP) (ICN#100878) to 10 ml TRAP bufferpH5.0 (100 mM Sodium Acetate, 11.5 mg/ml Sodium Tartrate (ICN#195503)).The media was aspirated off the assay plate and discarded. Cells werefixed for 1 minute with 1:1 ethanol and acetone solution and washed oncewith 100-150 μl/well of PBS (Hyclone#SH30256.02). 100 μl of TRAPsolution was added to each well, and the plate was incubated at 37° C.for 2 hours. The reaction was stopped by adding 50 μl/well of a 0.2NNaOH solution and the absorbance was read at 405 nm. If the absorbancereached saturation, the solutions were diluted 1:5 and read again.

[0244] Tartrate-Resistant Acid Phosphatase (TRAP) Cytological Staining

[0245] In parallel experiments, osteoclast formation from the RAW 264.7cells was measured by the presence of Tartrate-Resistant AcidPhosphatase (TRAP) multinucleated positive cells using cytologicalstaining. 0.1 M Acetate buffer was prepared prior to staining bycombining 35.2 ml of a 0.2M Sodium Acetate solution, 14.8 ml of a 0.2MAcetic Acid solution and 50 ml of water. 10 ml TRAP buffer pH5.0 (50 mMAcetate Buffer, 30 mM Sodium Tartrate (Sigma#S-8640), 0.1 mg/ml NaptholAS-MX Phosphate Disodium Salt (Sigma#N-5000), 0.1% Triton X-100) wasprepared fresh for each assay. The TRAP buffer was warmed in a 37° C.water bath and 0.3 mg/ml of Fast Red Violet LB Stain (Sigma#F-3381) wasadded and the stain was returned to the 37° C. water bath. The media wasaspirated off the assay plate and discarded. The cells were washed oncewith 150 μl/well of PBS (Hyclone#SH30256.02) and subsequently fixed with100 μl/well of a 10% Glutaraldehyde solution for 15 minutes at 37° C.The cells were washed twice with 150 μl/well of pre-warmed PBS. 100 μlof the pre-warmed TRAP stain was added to each well and the plate wasincubated at 37° C. for 5-10 minutes. The TRAP stain was removed and 100μl of PBS was added to each well to prevent cells from drying out. TRAPpositive multinucleated cells (more than three nuclei) were counted.

Example 2

[0246] Antagonistic RANKL Variant Library Design

[0247] Antagonistic RANKL variants were generated using thedominant-negative strategy which includes the single chain variety, andby the selection of competitive inhibitor antagonists.

[0248] Dominant-Negative RANKL Library Variants

[0249] PDA™ was utilized to generate a human structural model of RANKLin addition to selecting amino acids that disrupt RANK binding. The lackof a crystal structure for human OPGL necessitated the creation of ahomology model where PDA™ was used for side chain placement based on thehuman sequence and mouse RANKL structure (PDB# 1JTZ). This modeling wasfacilitated by the 87% sequence identity between the mouse and humanRANKL sequences. The dominant-negative RANKL therapeutic strategy isbased on the design of novel RANKL variants that have reduced receptorbinding and/or activation properties and the ability to heterotrimerizewith wild-type RANKL (FIG. 21). In other words, RANKL variants that donot activate RANK will exchange with wild-type RANKL protein andsequester it into inactive heterotrimers, inhibiting its activity. Thenumber and activity of osteoclasts will be lowered as a result of thistreatment leading to a decrease in bone resorption and an overallincrease in bone mineral density.

[0250] The dominant-negative RANKL variants were designed bysubstituting the amino acids at key RANKL-RANK contact points with aminoacids that disrupt the ability of the ligand to activate receptor. PDA™technology was used to select appropriate substitutions for optimalprotein folding. The exchange of these trimeric RANKL variants withtrimeric wild-type RANKL will result in the deactivation of thewild-type RANKL and reduced osteoclast formation. To help accomplishthis goal more effectively, the RANKL variants can also be designed topreferentially heterotrimerize with wild-type RANKL. The list of 89RANKL library variants is found in FIG. 22. Additional libraries basedon the combinatorial assembly of these variants and between thesevariants and the solubility variants (FIG. 2b) were also produced.

[0251] Single Chain Dominant-Negative Polypeptides

[0252] Multiple strategies for covalent linkage of monomers exist. Theseincluded, but are not limited to: polypeptide linkages between N andC-termini of two domains, made up of zero or more amino acids (resultingin single chain polypeptides comprising multiple domains); linkage via adisulfide bond between monomers; linkage via chemical crosslinkingagents.

[0253] Multiple strategies exist for modification of individual domainssuch that receptor binding is removed (or reduced). These include, butare not limited to: amino acid modifications that create stericrepulsion between ligand domain and receptor; modifications that createelectrostatic repulsion; modifications that create unfavorabledesolvation of amino acids; and chemical modification of amino acids atthe ligand/receptor interface (e.g. PEGylation or glycosylation).

[0254] Linkage of RANKL Monomers into a Trimer

[0255] To improve the dominant negative behavior of these mutants,single-chain dimers between two modified receptor interaction domainsare being created.

[0256] RANKL Variant Library Construction

[0257] The full-length RANKL cDNA was cloned using RT-PCR on lymph nodecDNAs. Two lengths of RANKL, amino acids 147 to 317 and 159 to 317, weresubcloned into pET33b after PCR-mediated addition of TEV and HIS tags(FIG. 1b).

[0258] Muatagenesis reactions were performed to introduce the nucleotidechanges that result in the desired amino acid substitutions. Themutagenesis reactions utilize pairs of divergent and overlappingoligonucleotides with nucleotide variations that introduce the specificamino acid changes. These oligonucleotides are extended using Pfupolymerase through multiple rounds of thermal cycling and theseextension products are transformed into TOP10 bacterial cells afterdigesting the products with DpnI. Clones were sequenced to confirm thedesigned changes and to insure no modifications were introduced into thecDNA during the thermal cycling.

[0259] RANKL Variant Screening for Non-Agonists, Superagonists, andAntagonists

[0260] A library of 89 human RANKL variants was constructed with allmembers of the library comprising the amino acid sequence in FIG. 1b andthe two solubility-imparting modifications, C221S/I247E in addition tothe 89 computationally designed inhibitory modifications from FIG. 22.These 89 human RANKL variant proteins were expressed and purified fromE. coli and 52 were screened for non-agonists, superagonists, andantagonists in an assay that monitors osteoclastogenesis in RAW264.7cells (FIGS. 23-30). Treating RAW264.7 cells with the purified humanRANKL variant protein and measuring the amount of TRAP releasedidentified non-agonist and superagonist variants (FIG. 29). Of the 52RANKL variants screened, 21 RANKL variants were identified to benon-agonists, while C221S/I247E/A172R was found to be a superagonist. Inaddition, the antagonism screens described in FIGS. 26-28 identified thesix antagonizing variants listed in FIG. 30. These antagonizing variantshave the ability to lower the osteoclastogenesis activity of humanRANKL.

[0261] Tartrate-Resistant Acid Phosphatase (TRAP) Activity Bioassay

[0262] The TRAP activity bioassay was used for assaying non-agonism,super-agonism, and antagonism. TRAP activity was measured after addingthe RANKL variant protein to cell lines such as RAW264.7 to identifyRANKL variants that are non-agonists or super-agonists (FIGS. 23-25,29). An additional screen was performed to identify antagonist RANKLvariants by mixing RANKL variant protein with one of the active RANKLsolubility variants, C221S/I247E, and adding the protein mixture to theRAW264.7 and measuring TRAP activity. Antagonist RANKL variants wereidentified that lowered the activity of the RANKL solubility variantC221S/I247E (FIGS. 26-28, 30).

[0263] RAW 264.7 cells (ATCC#TIB-71) were cultured and maintained in 75cm² flasks in 5% CO₂ humidified incubator at 37° C. in growth mediacontaining α-MEM w/2 mM L-Glutamine (Gibco-BRL#12571-063), 10% HeatInactivated Fetal Bovine Serum (FBS) (Hyclone#SH30071.03) andPenicillin/Streptomycin (Gibco-BRL#15140-122) 100 units/ml and 100 μg/mlrespectively.

[0264] Recombinant mouse RANKL (R&D#462-TR), recombinant human solubleRANKL (Biosource#PHP0034), and recombinant human soluble RANKL variantswere diluted to their working concentration in assay media consisting ofα-MEM w/I-glutamine (Gibco-BRL#12571-063) supplemented with 10% HeatInactivated Fetal Bovine Serum (FBS) (Hyclone#SH30071.03) andPenicillin/Streptomycin (Gibco-BRL#15140-122) 100 units/ml and 100 μg/mlrespectively. 250 μl of RANKL standards and samples were added to96-well assay plate in triplicate or quadruplicate, and serially diluted1:2 by transferring 125 μl into subsequent wells containing 125 μl ofassay media.

[0265] Aspirating the exhausted media, adding 10 ml of pre-warmed mediato the flask, and scraping cells from the flask using a cell scraperharvested the cells. The cells were transferred to a 15 ml conical tube,spun at 1000 rpm for 5 minutes and resuspended in 10 ml of assay media.The cells were counted and seeded at density of 1.5×10³/125 μl/well in96-well assay plate. Assay plates were incubated at 37° C. in 5% CO₂humidified incubator for 72 hours.

[0266] The amount of osteoclast-like cells induced from the RAW 264.7cells was determined by measuring the Tartrate-Resistant AcidPhosphatase (TRAP) activity. TRAP substrate was prepared by adding 20 mgof p-nitrophenol phosphate (pNPP) (ICN#100878) to 10 ml TRAP bufferpH5.0 (100 mM Sodium Acetate, 11.5 mg/ml Sodium Tartrate (ICN#195503)).The media was aspirated off the assay plate and discarded. Cells werefixed for 1 minute with 1:1 ethanol and acetone solution and washed oncewith 100-150 μl/well of PBS (Hyclone#SH30256.02). 100 μl of TRAPsolution was added to each well, and the plate was incubated at 37° C.for 2 hours. The reaction was stopped by adding 50 μl/well of a 0.2NNaOH solution and the absorbance was read at 405 nm. If the absorbancereached saturation, the solutions were diluted 1:5 and read again.

[0267] Tartrate-Resistant Acid Phosphatase (TRAP) Cytological Staining

[0268] In parallel experiments, osteoclast formation from the RAW 264.7cells was measured by the presence of Tartrate-Resistant AcidPhosphatase (TRAP) multinucleated positive cells using cytologicalstaining. 0.1M Acetate buffer was prepared prior to staining bycombining 35.2 ml of a 0.2M Sodium Acetate solution, 14.8 ml of a 0.2MAcetic Acid solution and 50 ml of water. 10 ml TRAP buffer pH5.0 (50 mMAcetate Buffer, 30 mM Sodium Tartrate (Sigma#S-8640), 0.1 mg/ml NaptholAS-MX Phosphate Disodium Salt (Sigma#N-5000), 0.1% Triton X-100) wasprepared fresh for each assay. The TRAP buffer was warmed in a 37° C.water bath and 0.3 mg/ml of Fast Red Violet LB Stain (Sigma#F-3381) wasadded and the stain was returned to the 37° C. water bath. The media wasaspirated off the assay plate and discarded. The cells were washed oncewith 150 μl/well of PBS (Hyclone#SH30256.02) and subsequently fixed with100 μl/well of a 10% Glutaraldehyde solution for 15 minutes at 37° C.The cells were washed twice with 150 μl/well of pre-warmed PBS. 100 μlof the pre-warmed TRAP stain was added to each well and the plate wasincubated at 37° C. for 5-10 minutes. The TRAP stain was removed and 100μl of PBS was added to each well to prevent cells from drying out. TRAPpositive multinucleated cells (more than three nuclei) were counted.

We claim:
 1. A variant RANKL monomer protein comprising a variant of anextracellular domain of a RANKL protein.
 2. A mixed trimer comprising atleast one variant RANKL monomer and at least one wild-type RANKLmonomer.
 3. A variant RANKL protein according to claim 1 wherein saidvariant RANKL protein comprises a modification that confers a reductionin hydrophobicity.
 4. A variant RANKL protein according to claim 1wherein said variant RANKL protein comprises a modification that confersan increase in polar character.
 5. A variant RANKL protein according toclaim 1 wherein said variant RANKL protein comprises a modification thatconfers a reduction in hydrophobicity and an increase in polarcharacter.
 6. A variant RANKL protein according to claim 1 wherein saidvariant RANKL protein comprises a modification of at least one positionselected from the group consisting of: T173, S183, I207, A232, and I247.7. A variant RANKL protein according to claim 6 wherein said variantRANKL protein comprises at least one modification selected from thegroup consisting of: A232K, I207K, I207R, I247E, I247K, I247Q, andI247R.
 8. A variant RANKL protein according to claims 1 wherein saidvariant RANKL protein comprises a modification from a cysteine residueto a non-cysteine residue.
 9. A variant RANKL protein according to claim8 wherein said variant RANKL protein comprises a modification at C221.10. A variant RANKL protein according to claim 9 wherein said variantRANKL protein comprises at least one modification selected from thegroup consisting of: C221A, C221S, C221T, and C221V.
 11. A mixed trimeraccording to claim 2 wherein said mixed trimer is substantiallyincapable of activating receptor signaling.
 12. A mixed trimer accordingto claim 2 wherein said mixed trimer will physically interact with areceptor interface at at least one receptor binding site to render saidreceptor substantially incapable of activating receptor signaling.
 13. Avariant RANKL protein according to claim 1, wherein said variant hasreduced affinity for a desired receptor as compared to wild type RANKLprotein and retains the ability to interact with other receptorinteraction domains.
 14. A variant RANKL protein according to claim 1,wherein said variant RANKL protein physically interacts with a naturallyoccurring RANKL protein to reduce the ability of the naturally occurringRANKL to activate at least one receptor.
 15. A variant RANKL proteinaccording to claims 1, wherein said variant RANKL protein comprises amodification at a receptor contact position.
 16. A variant RANKL proteinaccording to claim 1, wherein said variant RANKL protein comprises amodification at a trimer interface position.
 17. A variant RANKL proteinaccording to claim 1 wherein said variant RANKL protein comprises amodification at at least one position selected from the group consistingof: 172, 179, 180, 181, 182, 183, 187, 188, 189, 190, 191, 192, 193,222, 223, 224, 225, 226, 227, 228, 233, 234, 235, 236, 237, 238, 239,240, 241, 246, 247, 248, 249, 250, 251, 252, 253, 267, 268, 269, 270,284, and 297, 298, 299, 300, 301, and
 302. 18. A variant RANKL proteinaccording to claim 1 wherein said variant RANKL protein comprises amodification at at least one position selected from the group consistingof: 181, 190, 192, 223, 225, 226, 227, 237, 248, 249, 269, 270, and 298.19. A variant RANKL protein according to claim 1 wherein said variantRANKL protein comprises at least one modification selected from thegroup consisting of: K181Q, D190Q, G192A, R223Q, H225E, H225N, H225R,H225T, E226D, E226Q, E226R, T227Q, Q237E, K248E, I249R, E269K, E269Q,E269T, F270T, and K298E.
 20. A variant RANKL protein according to claimI wherein said variant RANKL protein comprises a modification at atleast one position selected from the group consisting of: 163, 165, 167,193, 195, 213, 215, 217, 219, 221, 235, 237, 239, 244, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 268, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 300, 302, 304, 305, 307, 311,313 and
 314. 21. A variant protein according to claim 1, wherein saidvariant protein is chemically modified.
 22. A variant protein accordingto claim 21, wherein said chemical modification is PEGylation.
 23. Avariant protein according to claim 21, wherein said chemicalmodification is glycosylation.
 24. A variant protein according to claim21, wherein said chemical modification is a fusion of said variantprotein to another entity.
 25. A variant protein according to claim 24,wherein two or more modified domains are covalently cross-linked.
 26. Avariant protein according to claims 25, wherein two or more RANKLvariant proteins are covalently linked by a linker peptide.
 27. Avariant protein according to claim 26, wherein said linker peptide is asequence of at least one and not more than about 30 amino acid residues.28. A variant protein according to claim 27, wherein said linker peptideis a sequence of at least 5 and not more than about 20 amino acidresidues.
 29. A variant protein according to claim 28, wherein saidlinker peptide is a sequence of at least 10 and not more than about 15amino acid residues.
 30. A variant protein according to claim 26,wherein the linker peptide comprises at least one of the following aminoacid residues: Gly, Ser, Ala, or Thr.
 31. A mixed trimer according toclaim 2, wherein said mixed trimer binds to the RANK receptor but doesnot substantially activate the RANK receptor.
 32. A variant RANKLprotein according to claim 31, wherein said variant RANKL proteincomprises a modification within a receptor contact domain.
 33. Arecombinant nucleic acid encoding the protein of claim
 1. 34. A hostcell comprising the recombinant nucleic acid of claim
 33. 35. A methodof producing a non-naturally occurring RANKL protein comprisingculturing the host cell of claim 34 under conditions suitable forexpression of said nucleic acid.
 36. A method according to claim 35,further comprising recovering said RANKL protein.
 37. A pharmaceuticalcomposition comprising a variant RANKL protein according to claim 1 anda pharmaceutical carrier.
 38. A method for treating a RANKL-relatedcondition comprising administration of a variant RANKL protein accordingto claim
 1. 39. A method of antagonizing a naturally occurring RANKLprotein comprising contacting a naturally occurring RANKL monomerprotein with a variant RANKL monomer protein comprising at least avariant extracellular domain of a RANKL protein, to form a mixed RANKLoligomer
 40. A method of making a mixed RANKL oligomer comprisingcontacting at least one variant RANKL protein comprising at least avariant extracellular domain of a RANKL monomer protein with ahomo-oligomer comprising naturally occurring RANKL monomer proteins,under conditions whereby at least one naturally occurring RANKL monomerexchanges with a variant monomer to form a mixed oligomer.